In recent years, the problem of antibiotic resistance has become increasingly severe worldwide. Antimicrobial peptides (AMPs), due to their safety and low propensity for developing resistance, are promising candidates as new antimicrobial drugs to replace antibiotics, attracting more and more research teams to conduct in-depth studies. However, there are currently no reports of successful biosynthesis of antimicrobial peptides. Our project focuses on the human antimicrobial peptide LL-37, dedicated to the efficient and high-yield biosynthesis of high-performance LL-37, and constructing a cell factory for short peptide synthesis.
We sincerely hope that our team's exploration in antimicrobial peptides and multifunctional short peptide synthesis can serve as a stepping stone for future iGEM teams. We are willing to share our experiences and results with everyone, hoping these experiences can help other teams.
The strength of promoters controls the expression level of target proteins. To improve LL-37 expression yield, we modified the promoter upstream of the LL-37 expression cassette, making two effective attempts. The first was replacing the GAL1 and GAL10 core promoters of the original GAL promoter with strong core promoters THF1 and GAP Promoter (BBa_2522RNSA and BBa_2516BSX2) , which showed slight improvement; the second was replacing the original UASGAL with triple tandem UASCLB2, which significantly enhanced the promoter(BBa_25CSLYKP).
We believe these two methods will provide new insights for iGEM teams interested in promoter engineering.
We connected an LL-37 expression cassette to both ends of the engineered GAL promoter, constructing a multi-copy site-specific integration plasmid (BBa_251COFY9), thereby greatly enhancing LL-37 expression yield.
The multi-copy genomic integration strategy provides an effective universal strategy for other teams to improve target product expression levels.
To explore the intracellular transport pathway of LL-37, we conducted fluorescence co-localization experiments. We selected six organelles and their corresponding targeting proteins: endoplasmic reticulum (CNE1), nucleus (NYV1), mitochondria (COX4), peroxisome matrix (PEX8), vacuole (PRC1), and cell membrane (SNC1), linked with red mCherry (BBa_25NO7917、BBa_25MJPWW6、BBa_25CZMSB9、BBa_25DXCMUY、BBa_25MJPWW6、BBa_25RWCL3T）, while constructing LL-37 linked with green eGFP（BBa_25X0UQIK). By merging the fluorescence images, we determined the distribution of LL-37.
We confirmed that the fluorescence co-localization method is reliable and effective, serving as a feasible and powerful tool for other iGEM teams to locate intracellular synthesis and transport sites of substances.
Based on the growth conditions and LL-37 synthesis yield of different S. cerevisiae strains, we initially selected the engineering strain CEN.PK 113-7D as the chassis cell for modification. Using homologous recombination, we mainly performed two types of gene knockout attempts on this strain's genome.
See Results page for details.
Our successfully constructed multiple gene knockout strains not only promise to broadly enhance heterologous expression yields of amino acids and peptides, but also demonstrate that knocking out specific genes to relieve dependence on specific inducers is an efficient metabolic engineering strategy.
Secretory expression of short peptides lacks the mature technical system available for large molecular proteins like enzymes. Due to their small molecular weight and the positive charge of our target protein LL-37 itself, we encountered many challenges during the project and devised solutions:
The challenges our team encountered and the corresponding solutions are common and universal in short peptide biosynthesis. Many unknown challenges remain, but our experience can help future iGEM teams working on short peptide synthesis avoid some difficulties, provide direction for constructing cell factories for short peptide synthesis, and hopefully help solve the antibiotic resistance problem, promoting sustainable global health development.
We established CytoGrow, a multi-component modelling system for optimizing Saccharomyces cerevisiae production of antimicrobial peptides:
1.1.1 Grow-Medium: Hybrid optimization combining quadratic response surface modelling with Gaussian process residuals
1.1.2 Grow-Yeast: Biomass Growth Kinetics Model
Enables continuous monitoring of S. cerevisiae biomass throughout fermentation cycles, transforming discrete experimental measurements into continuous predictive curves.
1.1.2 Grow-Yeast: Biomass Growth Kinetics Model
Tracks glucose depletion dynamics to predict the critical transition point when S. cerevisiae shifts from growth to LL-37 production phase (post-glucose exhaustion).
We developed CytoGuard, an innovative deep learning framework that significantly advances antimicrobial peptide (AMP) activity prediction. Unlike traditional methods, CytoGuard integrates multiple pre-trained protein language models (ESM-2, Ankh, ProtT5) with Hypergraph Neural Networks to capture complex sequence-structure relationships.
Key innovations:
We created CytoEvolve, the first framework to combine Discrete Diffusion Models with Reinforcement Learning for AMP optimization. This approach addresses the challenge of exploring vast sequence spaces while maintaining biological relevance.
Technical breakthroughs:
The model successfully produced two LL-37 variants showing stronger antimicrobial effects than the original sequence, demonstrating practical applicability.
We have made key components of our computational framework available on GitHub for the iGEM community. Currently, our repository includes:
We hope these computational tools will empower future teams to rapidly engineer peptides for diverse applications, from therapeutics to industrial enzymes, without extensive wet lab resources.
To boost engaging synthetic biology education— a key part of our iGEM project—we developed "Cytopia Defense", an online game to demystify antimicrobial peptides (AMPs) like LL-37. Players act as the "Antimicrobial Squad," strategically deploying LL-37 to fight pathogens. This turns abstract concepts into hands-on learning, helping users grasp not just AMP functions, but how to optimize them.
Launched during our outreach phase, it exceeded goals: over 3,000 cumulative visits and a 24-minute average playtime—surpassing typical educational content attention spans. Its feedback system collects player behavior data , which we use to inspire young interest in synthetic biology and refine our overall science communication. Ultimately, "Cytopia Defense" is more than an educational tool—it strengthens our iGEM project’s connection to the diverse communities we aim to educate.
Guided by our belief that science and art are universal languages for understanding the world— a core value of our iGEM outreach—we partnered with SKLBE-CHINA to launch the "Light of Life" bio-art initiative. This collaboration aimed to break disciplinary barriers and spark public, especially youth, interest in life sciences by turning abstract synthetic biology concepts into visual stories.
Instead of focusing on complex formulas, we invited participants to reimagine life science through painting: they transformed cells, antimicrobial peptides (central to our project), and DNA into imaginative artworks—such as the vivid "Cell City," where cellular structures became a bustling urban landscape, and "Antimicrobial Peptide Little Guards," personifying AMPs as protectors.
The initiative gained strong resonance, attracting participants of all ages from regions across 6 provinces including Jiangsu and Shandong—uniting audiences beyond traditional science circles in these areas. By translating technical principles (like how AMPs interact with cells) into colorful, narrative-driven art, we didn’t just showcase science’s beauty; we created an innovative bridge for reaching broader local and inter-provincial communities. This activity turned passive science learning into active creativity, not only reinforcing our iGEM goal of making synthetic biology accessible to diverse groups in these 6 provinces but also offering a replicable model for global synthetic biology outreach, inspiring more cross-regional and international efforts to demystify science through art.
Due to its technical complexity, synthetic biology is often subject to public misconceptions. To address this, we collaborated with 33 other university teams to author a brochure called "Smashing synthetic biology rumours science brochure" dedicated to debunking these common myths.
The handbook uses clear, real-life analogies and intuitive illustrations to transform complex concepts into easy-to-understand content. To tackle the most frequent misconceptions, it employs a structured format for each: Misconception Statement and Scientific Breakdown. This approach systematically responds to each myth and helps the public identify misinformation.
Ultimately, this handbook serves as both an informational guide to synthetic biology and a scientific tool for debunking myths, empowering the public to develop a rational perspective on this cutting-edge technology.
Recognizing that community residents hold diverse misconceptions about antimicrobials, we designed interactive activities tailored to different age groups to make our science communication more relatable and effective.
For families, we introduced the "Handwashing Song" and a game called "Cytopia Defense". These activities encouraged parents and children to sing and practice proper handwashing together, reinforcing the message that washing with running water and soap is the most effective method, and reliance on special antibacterial products is unnecessary. For the elderly and young children, we distributed illustrated brochures and used quiz-style questions—such as, "Should a child with a runny nose take two antibiotic pills to prevent pneumonia?"—to directly address and correct these cognitive biases.
Ultimately, our goal was to help residents distinguish between genuine and perceived needs for antimicrobials, empowering them to integrate scientific health practices into their daily lives.
To make the abstract principle of "precision targeting" in synthetic biology more understandable, we designed this interactive experiment. The frisbees in the participants' hands represent antimicrobial peptides (AMPs), while the markers on the field simulate "bacterial" pathogens.
Participants need to adjust their throwing force and angle to counteract minor environmental disturbances, aiming to land the frisbee precisely within the "bacteria" zone. This process is analogous to how an AMP locates its target within the body. Upon a successful hit, participants can intuitively grasp that "precision delivery" is not a matter of luck, but is achieved by assessing the target and controlling the process. This is just like optimizing an AMP delivery system to act precisely on pathogens while minimizing its impact on normal cells.
To make the abstract principle of "precision targeting" in synthetic biology more understandable, we designed this interactive challenge. The curling stones in the students' hands represent antimicrobial peptides, while the target area on the rink simulates the "lesion site."
Participants need to adjust their throwing force and trajectory to counteract minor disturbances on the ice, aiming to slide the stone precisely into the target zone. This process mirrors how antimicrobial peptides locate and act on pathogens within the body. When a stone lands accurately, students intuitively grasp that “precision delivery” is not a result of chance, but comes from assessing the target and controlling the delivery process. This is analogous to optimizing an antimicrobial peptide system to act precisely on harmful bacteria while minimizing impact on normal cells.