Our project has made significant contributions to the iGEM Registry, adding a series of related components and laying the groundwork for the subsequent work of other teams. Besides providing the basic parts, we also constructed, tested, and verified the composite part (BBa_257BU3C1) which demonstrated remarkable anti-fouling, adhesion, and antibacterial properties. It also provided theoretical and conceptual support for the future application of biological coating proteins in medical devices. Furthermore, during the testing process, we developed and adopted a series of testing methods to evaluate the adhesion, stain resistance and antibacterial properties of the coating proteins. These methods can also be referred to and utilized by the subsequent teams.

Based on the literature review, we selected a series of functional units related to the research objective, demonstrating the corresponding adhesion, antibacterial, and anti-fouling properties. These adhesion proteins were fused onto the pET21a-His vector for expression and purification. Including BBa_255Q1952 ，BBa_257N0R0R

In order to determine the activity of different antimicrobial peptides, we chose the synthetic method to evaluate their activity against Escherichia coli and Bacillus subtilis. At the same time, we mapped these antimicrobial peptides to the expression system of Escherichia coli and described their nucleic acid sequences, including BBa_25GXXDGP, BBa_25UMVS3P, BBa_257GOEC7，It can target the membrane structure of bacteria and exhibits broad-spectrum antibacterial activity. Meanwhile, based on the experimental results, we used the pET21a-His vector to perform fusion expression of different functional proteins BBa_257BU3C1，which including the anti-fouling peptide BBa_259UW0Q7. And evaluate the antibacterial activity, adhesion activity and anti-fouling activity of the fusion protein zwi-Mfp5-D51 respectively.

This project not only developed a multifunctional fusion protein coating with excellent adhesion, antibacterial, and antifouling properties but also established a comprehensive and systematic testing framework to evaluate its performance. By combining biochemical assays (such as NBT staining for hydroxylation), multiple adhesion evaluation methods (Coomassie Brilliant Blue staining and macroscopic pipette adhesion tests), and microbiological assessments (antibacterial activity and antifouling colony counting), the study provides a robust and multi-dimensional approach to thoroughly characterize coating functionality. This integrated testing strategy enhances the reliability and depth of coating assessment and can serve as a valuable reference for future research in biomedical coating development.