Developed a pipeline capable of predicting the secretion efficiency of secretory peptides

Our research aims to improve the secretion efficiency of chitinase in Escherichia coli. To achieve this, we utilized several bioinformatics tools and established a prediction pipeline that assessed the secretion performance of signal peptides based on secretion pathways and sequence features. Using over 2,000 signal peptide sequences, we tested the pipeline and validated its predictions in wet-lab experiments. This workflow enables future iGEM teams to efficiently identify high-performance signal peptides for fusion with their target proteins in E. coli, thereby enhancing protein secretion and improving experimental efficiency.

Developed a biosafety tool that uses protein similarity to find safety strains from the iGEM whitelist as substitutes for pathogenic bacteria in experiments

Since the iGEM whitelist does not include any Fusarium strains available to us, it has been difficult for us to verify whether the engineered bacteria we constructed have the potential to serve as a new type of biological agent for soybean root rot. To establish a laboratory system for verifying the antagonistic ability of engineered bacteria while complying with iGEM's official biosafety requirements, we developed a workflow. Based on protein structure similarity and homology, this algorithm can search whitelist organisms for proteins homologous to those from non-whitelist species, offering safe alternatives for experimental use, and provides a new approach for subsequent iGEM activities in the research on Fusarium spp. Using our tool, we found that the chitin synthase of Saccharomyces cerevisiae shares a certain degree of structural similarity with that of Fusarium, which can be used to validate the antagonism assay of β-amyrin in our project. The idea behind our algorithm is to provide an experimental reference for subsequent iGEM teams, helping them identify safety strains from the iGEM whitelist that are more similar to real pathogenic bacteria as substitutes, thereby ensuring the safe and effective implementation of experiments.

Developed a measurement tool based on safer antifungal indicator strains

Through a biosafety algorithm, we identified Saccharomyces Cerevisiae as a substitute for Fusarium spp. (the common pathogenic fungi causing root rot, which are not included in the official iGEM whitelist) for our experiments. However, no antagonistic effect was observed when our engineered bacterial strains were tested against Saccharomyces cerevisiae, which was inconsistent with the results of enzyme activity assays or docking. Our wet experiments have confirmed that chitinases can degrade chitin, and β-amyrin can target chitin synthase. We therefore hypothesized that this discrepancy may be related to the low proportion of chitin in the cell wall of Saccharomyces cerevisiae (only 1%)—even if all chitin in Saccharomyces cerevisiae were eliminated, it would not cause cell death.

To address the inability to determine the antagonistic effects of chitinases and β-amyrin, we aimed to modify the Saccharomyces cerevisiae system so that it could test the activity of our engineered bacteria. Literature research revealed that the cell wall of Saccharomyces cerevisiae is mainly composed of the following components (by dry weight): 30%-60% β-glucan, 20%-40% mannan, 10%-30% protein, 1%-2% chitin, and 5%-20% lipids (including phospholipids, ergosterol, etc.), along with trace amounts of inorganic salts, minerals, and other auxiliary components.

Snailase is a mixed enzyme extracted from the crop and digestive tract of snails, containing over 20 enzymes such as cellulase, hemicellulase, pectinase, amylase, decarboxylase, and protease. It can dissolve yeast cell walls and is widely used in cell biology and genetic engineering research. Thus, we hypothesized that pretreating Saccharomyces cerevisiae with Snailase might highlight the structural importance of chitin in the cell wall, enabling it to respond to chitinases and β-amyrin and thereby achieving the expected efficacy.

Our experimental results demonstrated that this sensitive Saccharomyces cerevisiae—specifically engineered to reduce the overall strength of its cell wall, thereby making the cell wall more sensitive to chitin content—can replace Fusarium spp., which are not listed on the official iGEM whitelist, for antagonism experiments. This modification, by weakening the cell wall’s structural integrity and heightening its responsiveness to chitin, not only facilitated the conduct of our experiments but also successfully resolved the issue faced by GEMS Taiwan in iGEM 2022—their failure to establish an antagonism system in yeast. Their previous challenge likely stemmed from using unmodified yeast, whose robust cell wall masked subtle antagonistic effects; in contrast, our strain’s reduced cell wall strength and enhanced chitin sensitivity make even mild interactions detectable.

Additionally, this engineered yeast system confirmed that both chitinases and β-amyrin exhibit antagonistic effects against yeast: chitinases, which target chitin, act more potently due to the cell wall’s increased sensitivity, while β-amyrin, with its cell wall-disrupting properties, benefits from the weakened structural barrier to exert stronger inhibitory effects.