Throughout our iGEM journey, our team has been dedicated to addressing critical challenges in infant nutrition. In doing so, we have not only developed innovative biological systems but also generated a wealth of knowledge and resources that we believe will be invaluable to future iGEM teams. Our contributions are multifaceted, spanning from the creation and detailed characterization of new BioBrick parts to the development of practical guides for experimental procedures, human practices, and scientific communication.

We have consolidated our experiences into a series of comprehensive documents and new data for the Parts Registry. These resources are designed to help future teams navigate similar challenges, avoid potential pitfalls, and build upon our work. Our contributions encompass four key areas: a collection of robustly characterized biological parts, insights from our research process, a detailed experimental manual, and effective strategies for event organization and outreach. We hope these efforts will empower future iGEMers to accelerate their projects and make an even greater impact.

The Bronze part we contributed to is β-Galactosidase, originally designed by Team iGEM07_USTC. In our project, we aimed to develop a biological system for producing functional β-galactosidase (lactase), the enzyme essential for lactose digestion. We chose to utilize the existing BioBrick part BBa_I732005, which contains the coding sequence for the lacZ gene from Escherichia coli K-12.

While the sequence for BBa_I732005 exists in the registry, its documentation lacks a complete, quantitative "gene-to-function" validation pipeline. We have filled this gap by performing a systematic, three-step characterization process, providing future iGEM teams with robust data and a clear protocol for expressing and validating a functional lactase enzyme.

1. Characterization Workflow

We performed a full workflow to validate this part, from initial gene cloning to final enzymatic activity quantification.

Step 1: Vector Construction and Verification

To express the enzyme, we first cloned the codon-optimized lacZ gene (from BBa_I732005) into a pET-28a expression vector. The successful construction of the pET-28a-lacZ plasmid was confirmed by agarose gel electrophoresis following restriction digestion. As shown in Figure 1, a distinct band was observed at the expected size of 3072 bp, which perfectly matches the length of the lacZ gene. This result validates the correct cloning of the part, providing the essential tool for protein expression.

Figure 1: Construction of BL21-LacZ. (A) The plasmid map of pET28a-LacZ; (B) The gene circuit of BL21-LacZ; (C) The flow chart of "Construction of BL21-LacZ"; (D) the agarose gel electrophoresis analysis of the lacZ gene fragment

Next, we confirmed the expression of the protein product. The pET-28a-lacZ plasmid was transformed into E. coli BL21(DE3), and protein expression was induced. Cell lysates were analyzed using SDS-PAGE and Western Blot. The Western Blot analysis (Figure 2), using an anti-His antibody, detected a single, prominent band at the expected molecular weight of ~116 kDa. This band was present exclusively in the lysate from the lacZ-engineered strain and absent in the control, providing unequivocal proof of successful and specific expression of the full-length lacZ protein.

Figure 2: (A) This is a flow chart of Western Blot; (B) SDS-PAGE analysis of recombinant lacZ overexpression in E. coli

The final and most critical step was to quantify the enzyme's catalytic activity. The expressed β-galactosidase was purified and added to a 10 mM lactose reaction system. The degradation of lactose was monitored over an 8-hour period by measuring the production of glucose using a glucose assay kit. The results (Figure 3) clearly demonstrate that the enzyme is highly active. The initial lactose concentration of 10 mM was steadily reduced to approximately 2.0 ± 0.4 mM after 8 hours, corresponding to an 80% degradation of the initial substrate.

Figure 3: (A)This is a flow chart of measurement of the lactose content lacZ breaks down; (B) Efficacy of recombinant β-galactosidase in lactose degradation

Value to the iGEM Community

By providing this comprehensive, quantitative characterization, we have significantly enhanced the value and reliability of BBa_I732005. Future iGEM teams can now use this part with greater confidence, supported by:

Verified Construction: Evidence that the part can be successfully cloned into a standard expression vector. Confirmed Expression: A clear Western Blot result demonstrating high-level expression of the full-length protein. Quantitative Functional Data: A robust in vitro assay showing that the expressed enzyme is highly active, with 80% lactose degradation in 8 hours.

This complete dataset transforms BBa_I732005 from a simple coding sequence into a well-documented, functionally validated tool, ready for use in projects requiring lactose hydrolysis, such as the production of lactose-free dairy products, biosensor development, or metabolic engineering applications.

2. Experimental Protocols and Troubleshooting

To ensure the reproducibility of our work, we are providing future teams with our detailed, step-by-step protocols for protein expression, purification, and the functional activity assay on our wiki's Experiments page.

Troubleshooting Tip: The lacZ gene is relatively large (~3.1 kb), which can sometimes pose challenges during cloning or lead to lower plasmid yields. We recommend using a high-fidelity polymerase for PCR amplification and ensuring the quality of the vector backbone preparation. For protein expression, inducing at a lower temperature (e.g., 16-20°C) for a longer period (16-20 hours) can improve the yield of soluble, active protein, although our experiments showed high activity even with standard induction protocols.

Effective communication is paramount in conveying the complexity and innovation of a synthetic biology project. Recognizing this, our team placed a strong emphasis on creating clear, professional, and impactful scientific illustrations. We utilized BioRender to design crucial project visuals, from intricate pathway flowcharts to the final results figures.

Through this process, we gained extensive hands-on experience and identified a need for a consolidated resource to help other researchers master this powerful tool. To contribute to the iGEM community and beyond, we have channeled our expertise into producing a comprehensive 36-page user guidance manual.

This manual is more than just a summary of our work; it is a step-by-step guide designed to flatten the learning curve for new users while offering valuable tips for seasoned illustrators. It covers everything from the foundational interface and workflow procedures to advanced features like collaborative editing, creating publication-quality graphics, and even importing protein structures directly from the PDB. Our goal is to equip future iGEM teams with the skills to elevate their scientific storytelling, ensuring their hard work is presented with the clarity and professionalism it deserves.

Document 1: BioRender User Guidance(Due to upload restrictions, we have divided the file into three parts.)

To prevent future teams from encountering the same obstacles we faced, we have compiled a detailed Experimental Manual. This document includes step-by-step protocols, lists of required materials, and, most importantly, a troubleshooting section that addresses common errors and provides solutions. We believe this resource will help increase the efficiency and success rate of experiments for teams working on similar projects.

Furthermore, our team has dedicated significant effort to creating clear and professional scientific illustrations. We have compiled our experiences and tips into a BioRender User Guidance document. This guide covers best practices for creating flowcharts, genetic circuit diagrams, and results figures, which will aid other teams in effectively communicating their scientific work.

Figures 4: Experimental Manual

Figures 5: Team members conducting interview in park

Effective communication and organization are key to a successful Human Practices component. Based on our experience conducting interviews and educational events, we have summarized several key strategies:

Define Roles and Plan Thoroughly: Clearly define the purpose of each activity and assign roles based on team members' strengths. For example, meticulous planners can write interview scripts, while outgoing members can lead public interactions. Communicate Clearly and Build Rapport: When engaging with the public, always start with a clear and concise introduction of your team and project. We found that offering a small, handmade gift created a friendly atmosphere and significantly increased willingness to participate. Be Open to Unexpected Opportunities: Our outreach efforts led to an invitation to present at a breastfeeding promotion exhibition, an opportunity we had not anticipated. Be flexible and ready to embrace new avenues for engagement. Review and Iterate: After each event, conduct a thorough review. Analyze feedback, assess whether you achieved your goals, and identify areas for improvement. This iterative process ensures continuous progress and enhances the impact of future activities.

Figure 6：Event organization tips

Source research experience

Gathering credible information is the foundation of any successful scientific project. We learned that a thorough and systematic approach is crucial. For future teams, we recommend the following:

Prioritize Reliable Platforms: Utilize academic search engines like Google Scholar, JSTOR, and NCBI (National Center for Biotechnology Information). Focus on recent peer-reviewed journal articles (ideally within the last decade). Verify Sources: Be cautious with user-editable platforms like Wikipedia. Always trace information back to its original source. Credible sources include publications from reputable researchers, official university or institutional websites, and established scientific journals. Use AI as a Helper, Not a Source: Artificial intelligence can be a powerful tool for brainstorming and summarizing, but it should never be treated as a primary source. AI-generated information can be outdated, inaccurate, or even fabricated. Always verify any claims or sources provided by AI using the reliable platforms mentioned above.

Figure 7: A guide to identifying credible and non-credible sources