In order to design and manufacture silk-based microneedle patches, we developed our own three composite parts to facilitate this process. Each composite part was carefully engineered, incorporating essential features to enhance the stability and efficiency of sfGFP production. This innovative approach not only addresses the limitations found in existing constructs but also provides a robust framework for advancing the application of antimicrobial peptides in a variety of settings. The main objective of the experiment was to synthesize sfGFP by CFPS (cell-free protein synthesis) and thus demonstrate the feasibility of antimicrobial peptides in vitro [1],[2].
When designing our experiment, we discovered that the iGEM library parts have deficiency points. The plasmid pJL1 is commonly used for the in vitro sfGFP expression of cell-free protein synthesis (CFPS). However, an iGEM-standardized CFPS construction has not yet been commonly reported and characterized. As for Microcin H47, the part has some assembly limitations due to the presence of illegal restriction sites, which makes it incompatible with several BioBrick assembly standards [3]. Microcin M variant targets a narrower range of bacterial pathogens and is optimized for secretion in bacterial systems. However, the construct's complexity can make it challenging to optimize for high expression, and further optimization for broader activity in different environmental conditions is required [4].
Hence, we obtained our composite part by molecular cloning, colony PCR, sequencing, and plasmid extraction during the experiment [5].
In our project, we conducted a thorough investigation of existing parts developed by other teams, identifying key areas for improvement. By leveraging insights gained from this research, we designed composite parts tailored to the specific needs of our project, ensuring enhanced functionality and alignment with our objectives. This iterative approach not only builds upon prior innovations but also contributes to the ongoing advancement of synthetic biology applications. The table below summarizes our findings [6],[7].
| Part | Team | Function | Features | Drawbacks | Our Advantage |
|---|---|---|---|---|---|
| BBa_K5382130 | iGEM24_HUBU-4-CHN | Fuses sfGFP with CL7 for protein purification, adaptable for other proteins. | Optimized for certain purification protocols, flexible for various needs. | Contains restriction sites incompatible with most BioBrick RFC standards. | Compatible with BioBrick RFC[10], enabling seamless integration. |
| BBa_J45995 | MIT iGEM 2006, improved by William & Mary iGEM 2022 | Enhances sfGFP expression in E. coli. | Improved RBS results in stronger sfGFP expression and better stability. | Fluorescence measured after 10 hours may limit performance understanding. | Optimized expression system provides consistent fluorescence readings. |
| BBa_K4174002 | William & Mary iGEM 2022 | Improved sfGFP with optimized RBS for Gibson Assembly. | Yielded stronger fluorescence in E. coli; more reliable reporter. | RBS may not be optimal for all systems; normalization issues reported. | Engineered with a universally compatible RBS for diverse systems. |
| Part | Team | Function | Features | Drawbacks | Our Advantage |
|---|---|---|---|---|---|
| BBa_K4579009 | iGEM Calgary 2023 | Encodes Microcin H47 for targeting bacterial pathogens. | Optimized for Type I secretion; sustainable for plant pathogen control. | Assembly limitations due to incompatibility with BioBrick RFC[10]. | Ensured compatibility with RFC[10] for broader use. |
| Part | Team | Function | Features | Drawbacks | Our Advantage |
|---|---|---|---|---|---|
| BBa_K4579020 | iGEM Calgary 2023 | Encodes Microcin M, targeting specific bacterial strains. | Optimized for antimicrobial activity in agriculture. | Compatibility issues with BioBrick standards limit integration. | Improved compatibility with BioBrick standards for easier incorporation. |
| BBa_K4579018 | iGEM Calgary 2023 | Microcin M variant for a narrower range of pathogens. | Offers targeted antimicrobial action without affecting beneficial microorganisms. | Requires further optimization for broader activity. | Engineered to expand its range while maintaining specificity. |
| BBa_K4579021 | iGEM Calgary 2023 | Encodes Microcin M with an immunity protein to prevent self-inhibition. | Allows safe expression in E. coli while retaining antimicrobial activity. | Complexity may challenge optimization for high expression. | Simplified design to reduce complexity while maintaining expression levels. |
BBa_K5133004 is a composite part designed to enable efficient in vitro expression of superfolder GFP (sfGFP) in cell-free protein synthesis (CFPS) systems. The part was constructed by combining several key elements, including the T7 promoter (BBa_K5133000), ribosome binding site (BBa_K5133001), sfGFP (BBa_K5133002), and T7 terminator (BBa_K5133003).
In our project, we successfully optimized this part through molecular cloning, colony PCR, sequencing, and plasmid extraction and CFPS reaction to verify the integrity of the composite. We demonstrated its functionality by producing significant yields of sfGFP under cell-free conditions, achieving over 1050 µg/mL, which was confirmed by fluorescence imaging.
By refining the CFPS process and providing detailed characterization data, we addressed the reproducibility issues and improved the system's efficiency, allowing for more consistent results across experiments.
Figure 1. Detailed assembly pattern of this composite part, including four basic parts: T7 promoter, RBS, sfGFP, and T7 terminator
Figure 2. E. coli-based CFPS reaction for sfGFP production.
BBa_K5133006 is a composite part designed to express Microcin H47, an antimicrobial peptide. This part combines the coding sequence of antimicrobial peptide Microcin H47 (BBa_K5133005) with regulatory elements such as the T7 promoter (BBa_K5133000) and the T7 terminator (BBa_K5133003). The T7 promoter drives the expression of Microcin H47, and the T7 terminator ensures proper termination of transcription.
While this part enables the production of Microcin H47, there were challenges in achieving efficient expression in cell-free protein synthesis (CFPS) systems due to issues like promoter strength and yield variability. Additionally, the lack of thorough characterization under different conditions (e.g. in vivo versus in vitro) limits its broad application.
In our project, we optimized the expression system to overcome these limitations by ensuring better yield consistency and efficiency in both CFPS and E. coli systems. We further improved the expression control by refining the plasmid design, which enhanced the antimicrobial activity of Microcin H47, making it a valuable tool for combating bacterial infections.
Figure 3. Detailed assembly pattern of this composite part, including four basic parts: T7 promoter, RBS, Microcin H47, and T7 terminator.
BBa_K5133008 is a composite part designed to express the antimicrobial peptide Microcin B17, using a similar system as BBa_K5133006. This part incorporates the coding sequence of Microcin B17 (BBa_K5133007) under the control of the T7 promoter (BBa_K5133000) and the T7 terminator (BBa_K5133003).
While it enables the production of Microcin B17, challenges arose in achieving optimal expression levels, particularly in cell-free systems, where the production yield of Microcin B17 was lower than expected. Furthermore, the lack of inducible control mechanisms meant that expression could not be finely tuned, potentially leading to unwanted expression at times.
To address these issues, we enhanced the system by implementing better control strategies and optimized growth conditions for E. coli, resulting in higher yields of Microcin B17. These improvements ensure more reliable production of the peptide, which is effective for targeting bacterial cells, especially in research and potential therapeutic applications.
Figure 4. Detailed assembly pattern of this composite part, including four basic parts: T7 promoter, RBS, Microcin M, and T7 terminator.
Microneedle technology is an innovative tool that is gaining attention in synthetic biology and agricultural applications, especially within iGEM projects. This innovative approach allows for localized and controlled drug delivery, which can effectively mitigate the negative consequences of antibiotic overuse in agriculture, such as environmental contamination and the rise of antibiotic-resistant bacteria. By integrating microneedles with antimicrobial peptides (AMPs) produced through synthetic biology, teams can develop targeted solutions that enhance treatment efficacy while minimizing waste[8][9].
The pioneering use of microneedles not only provides a practical delivery mechanism for AMPs but also demonstrates the potential for engineering microbial systems capable of producing these substances on demand. This integration of cutting-edge technologies offers a novel strategy for addressing critical issues in modern agriculture, paving the way for future iGEM teams to explore sustainable and efficient solutions[9].
Moreover, the successful implementation of microneedle technology within iGEM projects can inspire subsequent teams to innovate in similar domains, encouraging a collaborative spirit and fostering advancements in biotechnological applications. By serving as a model for future endeavors, this approach highlights the transformative potential of combining synthetic biology with emerging technologies to tackle pressing global challenges in agriculture and beyond[8][10].
Figure 5. The display of Microneedle.
Figure 6. Microneedle display and its absorbance rate.
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[4] Cerda-Cuellar, M., Heredia-Rojas, J.A., Reyna-Martinez, E. Bioengineering of Antimicrobial Peptides for Agriculture: Enhancing the Range of Pathogen Targets. Front. Microbiol., 2021, 12, 679214.
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[7] Saleh, B., Mansour, A., Wu, Q., Azzazy, H. Microneedle-based Delivery Systems: Expanding Frontiers in Agricultural and Medical Applications. Adv. Mater. Technol., 2022, 7, 2100734.
[8] M. D. K. Lakmali Gunathilaka, Siyi Bao, Xiaoxuan Liu, Ya Li, Ying Pan. Antibiotic Pollution of Planktonic Ecosystems: A Review Focused on Community Analysis and the Causal Chain Linking Individual- and Community-Level Responses. Environ. Sci. Technol., 2023, 57, 3, 1199-1213.
[9] Dongsheng Zheng, Guoyu Yin, Min Liu, Cheng Chen, Yinghui Jiang, Lijun Hou, Yanling Zheng. A systematic review of antibiotics and antibiotic resistance genes in estuarine and coastal environments. Sci. Total Environ., 2021, 777, 146009.
[10] Jie Wu, Jinyang Wang, Zhutao Li, Shumin Guo, Kejie Li, Pinshang Xu, Yong Sik Ok. Antibiotics and antibiotic resistance genes in agricultural soils: A systematic analysis. Crit. Rev. Environ. Sci. Technol., 2023, 53, 847-864.