Our project innovatively uses ROS as a biological switch, showing significant innovation and application potential, with the Registry ID BBa_K5332001. As a ubiquitous intracellular signaling molecule, ROS can sensitively respond to environmental stimuli, such as oxidative stress and pathogen invasion. Using it as a biological switch can provide engineered bacteria with a rapid and precise response mechanism, enabling it to activate or deactivate specific functions under certain conditions, such as secreting antimicrobial peptides or expressing reporter genes. This strategy not only expands the application scope of ROS but also offers new insights for developing intelligent engineered bacteria.
Our project successfully designed and developed a novel CMC adhesion protein with a unique glucan-binding domain, enabling targeted adhesion to the gut and probiotics. By leveraging this domain's specific binding to probiotics within the gut microbiome, we achieved precise targeted delivery. Additionally, our engineered protein enhances the colonization efficiency of probiotics in the gut, promoting better probiotic effects. The CMC protein can serve as a novel probiotic adhesion carrier, improving the stability and bioavailability of probiotics. By enhancing probiotic colonization, it helps regulate gut microbiota balance and improve gut health. We have added this component to the Registry as a new part with the ID BBa_K5332002.
Our iGEM project successfully integrated the melittin component into the engineered bacterial plasmid, with the Registry ID BBa_K5332003. Notably, although there are updated sequences in the Registry, we have refined and optimized the melittin peptide to further enhance its performance. Specifically, we connected two melittin monomers with a linker to form a hairpin structure. This modification effectively reduces the peptide's cytotoxicity, making it safer for use, while also enhancing its immune-stimulatory properties and improving its potential in inflammation regulation. By utilizing engineered bacteria to express melittin in the gut, we achieved effective suppression of intestinal inflammation. Once colonized in the gut, the bacteria continuously secrete melittin, enabling in situ treatment of inflammation. This innovative approach provides new strategies and methods for treating intestinal inflammation. Importantly, melittin, as a natural anti-inflammatory agent, effectively reduces reliance on antibiotics, thereby lowering the risk of antibiotic resistance.
We also integrated several functional modules into a single plasmid, showcasing the advantages of modular design. This approach enhances the system's flexibility and scalability, providing a valuable reference for future teams. On the pET-28a(+) plasmid, we integrated three functional elements: an anti-inflammatory factor (Mel), an adhesion factor (CMC), and a reactive oxygen species promoter (P[OSR]). This successfully constructed a multifunctional plasmid capable of simultaneously regulating the expression of multiple genes. For example, it can initiate the expression of anti-inflammatory and adhesion factors under oxidative stress, thereby endowing engineered bacteria with greater environmental adaptability and functional diversity.
Our project stands out in interdisciplinary integration and modular design, and we hope these methods can inspire future teams. Future projects could further optimize the ROS response system by selecting more sensitive ROS receptors or optimizing promoter sequences to enhance the sensitivity and response efficiency of engineered bacteria to ROS. Additionally, integrating more bioactive proteins with the ROS response system could be explored. Constructing more complex gene regulatory networks to achieve finer control over engineered bacteria, allowing them to respond to multiple environmental signals, could lead to the development of more robust engineered bacteria. Our plasmid is registered with ID BBa_K5332000 in the Registry, and we invite you to explore our carefully designed plasmid!