In our project, we focused on improving the secretion of surfactin using Bacillus subtilis as a model organism. Although previous iGEM teams have explored this approach for addressing petroleum pollution, we introduced an innovative method: overexpressing SecA, FtsE, and FtsY. These proteins are integral components of the Sec SRP transport pathway, which facilitates the translocation of proteins from the cytoplasm to the extracellular environment.
This contribution holds potential for future iGEM teams facing similar challenges in boosting protein secretion. By adopting our strategy of enhancing secretion pathways, teams can build upon our work to improve the efficiency of protein-based systems in various applications, including environmental and industrial biotechnology.
We combined plant bioremediation with bioengineered bacteria, presenting an integrated approach to addressing petroleum contamination. Plants can absorb and break down petroleum compounds while releasing root exudates that support microbial growth, enhancing oil removal. Meanwhile, microorganisms can stimulate plant growth, creating a mutually beneficial system that maximizes bioremediation efficiency.
This creative combination could inspire future teams to explore multi-organism systems, broadening the scope of environmental projects and stimulating innovation in project design. For example, future iGEM projects could further explore symbiotic relationships between various organisms to tackle complex environmental challenges.
During our human practices (HP) activities, we encountered several challenges but developed efficient strategies to overcome them. We hope these approaches will benefit future teams:
Engaging Experts and Communities: We actively reached out to experts and stakeholders, an essential practice that any future team can implement. Building relationships with external experts ensures the relevance and impact of iGEM projects.
Effective Interview Techniques: Before interviews, we prepared clear, concise questions. We recommend future teams do the same, ensuring the interview objectives align with project goals. Listening attentively and asking follow-up questions also maximized the quality of the data collected.
Questionnaire Design: Our approach to designing questionnaires included focusing on clarity, distributing them via appropriate channels, and using statistical tools for precise results. This methodological rigor in gathering feedback and data could be a valuable model for teams tackling complex societal issues.
Our experience in team management provided valuable lessons that can be applied by future teams:
Understanding Team Dynamics: We ensured that team roles aligned with each member’s strengths and capabilities. Future teams can adopt this approach to optimize performance and maintain a balanced workload across members.
Goal Setting and Execution: Clear, achievable goals were set at both the team-wide and sub-group levels. We used mind maps to visually represent tasks and responsibilities, ensuring clarity and preventing miscommunication. This approach could help future teams avoid confusion and stay on track throughout their projects.
Leadership Development: We placed significant emphasis on fostering leadership within the team. Assigning group leaders and setting clear expectations for meetings are leadership principles that can help maintain momentum and resolve issues efficiently.
Team Building: We encouraged cross-training between different team roles, ensuring that every member had a holistic understanding of the project. This not only improved team cohesion but also prepared team members to handle tasks beyond their initial roles, a valuable practice for any iGEM team.
We introduced creative and hands-on educational activities to engage the community in scientific learning:
Assembling Water Purifiers: In our educational outreach, we demonstrated water purification techniques by allowing participants to assemble water purifiers themselves. This hands-on approach helped solidify their understanding of environmental science and the importance of sustainability.
Seed Bag Activity: We distributed seed bags and accompanying educational materials to encourage gardening and sustainable practices. This activity allowed participants to experience plant growth first-hand, reinforcing concepts related to ecology and environmental stewardship.
Simplified Scientific Concepts: We created cartoon representations of complex scientific concepts, like the surfactin production pathway, to make them more accessible to a younger audience. Future iGEM teams can use similar methods to engage and educate diverse audiences in a fun and effective manner.
Objective and Methods
The objective of this experiment was to measure the laccase (BsLac) activity in crude enzyme extracts from engineered E. coli strains to verify successful expression and catalytic function.
1. Culture and Extraction:
The engineered strains were cultured overnight at 37°C. After centrifuging 2 mL of the culture at 8000 rpm for 10 minutes, the cell pellet was resuspended in PBS (pH 7.4) and disrupted by ultrasonication to obtain crude enzyme extracts.
2. Laccase Activity Assay:
Laccase activity was measured using the ABTS oxidation method. The reaction mix included 930 μL Na₂HPO₄-citric acid buffer (50 mM, pH 3.2), 20 μL ABTS (10 mM), and 50 μL enzyme extract. Absorbance at 420 nm was recorded for 1 minute to calculate enzyme activity.
3. Reaction Principle:
ABTS stock solution is deep blue, which becomes blue-purple upon dilution. When oxidized by laccase, the solution changes from blue to green or blue-green, with strong absorbance at 420 nm.
Results and Conclusion
The engineered strain (BL21-pET23b-BsLac) showed significantly higher absorbance at 420 nm compared to control strains (BL21 and BL21-pET23b), confirming active BsLac expression. The higher laccase activity in the engineered strain suggests efficient oxidation of ABTS, as evidenced by the color change to green/blue-green.This indicates that the BsLac strain has strong laccase activity, supporting its potential for environmental applications like oil degradation.
The BsLac-engineered strain demonstrated significantly enhanced laccase activity, confirming its ability to oxidize ABTS efficiently, and highlighting its application potential in environmental remediation.
Objective and Methods
The aim of this experiment was to determine the optimal pH and temperature for laccase (BsLac) activity, providing insights for its application. Laccase activity was measured at 30°C across a pH range of 2.0 to 6.0, using glycine-HCl buffer for pH 2.0 and Na₂HPO₄-citric acid buffer for pH 3.0–6.0. To assess the temperature effect, activity was measured from 20°C to 60°C in Na₂HPO₄-citric acid buffer (pH 3.2), using ABTS as the substrate.
Results and Conclusion
Laccase activity was highest at pH 3.2 and peaked at 40°C. Activity decreased significantly at lower and higher pH values, and temperatures above 40°C caused a marked decline, indicating enzyme instability. These findings highlight that BsLac is most active under moderately acidic conditions and at 40°C, guiding its potential use in biotechnological applications.
Objective and Methods
The aim of this experiment was to validate the laccase activity of engineered E. coli strains expressing laccase (BsLac) on the cell surface using cell surface display technology. Previously, only crude enzyme extracts from intracellular expression were tested. However, for real-world applications, such as biodegradation, surface-displayed enzymes are essential for effective function in live bacterial systems. In this experiment, ABTS was used as a substrate to measure the activity of live engineered strains. The reaction mixture consisted of 1 mL of 5 mM ABTS, 0.5 mL of cell suspension, and 1.5 mL of 0.1 M acetate buffer (pH 5.0). The samples were incubated at 30°C in the dark, and laccase activity was measured at 420 nm using spectrophotometry. Enzyme activity (U) was defined as the amount of enzyme required to oxidize 1 μmol of ABTS per minute, calculated per gram of bacterial dry weight.
Results and Conclusion
The results showed that the engineered strain BL21-pET23b-BsLac with surface-displayed laccase had significantly higher laccase activity compared to the control strain (BL21) and the strain expressing laccase intracellularly (BL21-pET23b). Additionally, the fusion strain (BL21-pET23b-INP-BsLac), which further enhances enzyme display on the cell surface, exhibited the highest activity, suggesting an improvement in the efficiency of laccase activity when displayed on the bacterial surface. These findings confirm that surface-displayed BsLac in live engineered strains can be a powerful tool for applications such as bioremediation. As shown in the image above, the experimental setup and results for the 12 samples (four groups with three replicates each) demonstrate the outcomes clearly.
Our contributions offer multiple pathways for future iGEM teams to build upon, from enhanced protein secretion methods to integrated bioengineering solutions and team management strategies. By sharing our innovations and lessons learned, we aim to empower the iGEM community to continue pushing the boundaries of synthetic biology and global impact.
