Parts
ε-PLL production parts
In this project, we utilized multiple plasmids and genetic parts to achieve efficient ε-PLL generation and performed two cycles of experiments aimed at optimizing ε-PLL synthesis in Bacillus subtilis. Initially, we introduced the pls gene from Streptomyces albulus into our engineered strain via the pMA5 plasmid. This gene encodes the enzyme ε-Poly-L-lysine synthetase (PLS), which catalyzes the polymerization of L-lysine monomers into a homopolymer of ε-poly-L-lysine. This unique enzyme utilizes ATP to directly link lysine molecules via special ε-peptide bonds, creating long chains of lysine that are then secreted outside the bacterial cell. The presence of the pls gene is therefore central to the successful production of ε-polylysine.
After confirming the presence of the gene, we measured the concentration of ε-PLL using spectrophotometric methods. However, we encountered an issue with production efficiency, as the concentration of ε-PLL was still too low to meet the demands of our project. To address this challenge, we designed a second experimental cycle focused on boosting the ε-PLL yield through two strategic modifications. First, we incorporated the PPK2 gene, which plays a critical role in ATP regeneration by facilitating the breakdown of polyphosphate, thereby increasing available ATP for more efficient polymerization. Second, we replaced the thrD gene in Bacillus subtilis with the lysC311 gene, which alleviates the natural inhibition of L-lysine production in Bacillus subtilis, ensuring a higher supply of this crucial precursor for ε-PLLsynthesis.
Through these two enhancements, we successfully increased the efficiency of energy utilization and L-lysine production, which in turn led to a significant optimization of ε-PLL synthesis. The result was a substantial improvement in both the yield and efficacy of the process, allowing us to achieve a more efficient and scalable production of ε-PLL
Biocontainment parts
Another significant contribution of our experimental design is the suicide switch system, which is crucial for management of our engineered strain and ensurance of the biosafety of ε-PLL production. In this project, we developed a nutrient-deficient suicide switch that leverages the alrA gene and a lac operon system positioned at its upstream. The alrA gene in Bacillus subtilis plays a critical role in converting L-alanine to D-alanine, a vital precursor required for the synthesis of bacterial cell wall peptidoglycan. This metabolic pathway is essential for the survival and integrity of the bacterial cell.
To render the natural Bacillus subtilis strain dependent on external conditions for survival, we first knocked out the alrA gene, which made the strain unable to build its cell wall. As a result, the engineered strain could not survive on its own without external intervention. Following this, we reintroduced the alrA gene and we incorporated a lac operon system upstream of the gene.
This strategic design effectively placed the strain under our full control, since under these conditions, the strain would only be able to survive if the plasmid remained inside the cell, and if an external inducer, lactose or IPTG, was provided. The presence of lactose would activate the lac operon, leading to the expression of the alrA gene.
By constructing this comprehensive biocontainment system based on the alrA gene and lac operon, we have successfully ensured the biosafety of ε-PLL production. This suicide switch not only prevents uncontrolled growth of the engineered strain in unintended environments but also provides a robust and effective safeguard, allowing us to confidently scale up the ε-PLL production process while maintaining strict control over the engineered bacteria.
Efficient filtration device
To contribute to future iGEM teams, we developed a user-friendly purification device designed for efficient extraction of ε-PLL from the bacterial culture supernatant of our engineered strain. This hardware system demonstrates a continuous downstream purification workflow that simplifies and streamlines the entire process. By integrating a column-based filtration method, our device enables convenient and productive ε-PLL purification, providing an accessible approach for others working with similar biopolymer products. Additionally, the eluent obtained from this system can be directly applied in cling film production, showcasing the continuity between different stages of our project. Beyond its immediate application, this design offers future IGEM team a practical framework that bridges the dry lab and wet lab, facilitating a more seamless transition from bioproduction to material utilization for future synthetic biology projects. In addition, providing a column filtration process for product obtaining from engineered strains.
Film formation Protocols
We tested three biosynthetic film containing ε-PLL and tried to find the one with the best mechanical and antibacterial ability. After several tests, we chose a sodium alginate-chitosan bilayer protocol since it embodied a relatively excellent effect. Then we started to design and conduct plenty of experiments to obtain the optimal conditions for our film. To enhance our film's mechanical performance, we sprayed calcium chloride as plasticizers to increase cross-linking effect (For details please refer to experiments-protocol), aiming to improve the mechanical performance of out film; we constantly adjusted variables that influence the performance including the amount of ε-PLL added, the concentration of calsium cloride and drying time in order to find a balance between feasibility and integrated performance. Eventually, we confirmed the optimal conditions and produced film with ε-PLL as our final product. This protocol provides an easy and robust way to make a double layer film at a relatively low cost.
Bio-UNO card
Based on our comprehensive human practices work and business plan development, our team has made valuable contributions to the iGEM community by creating and sharing a series of practical resources and strategic insights. We have developed and validated an educational card game based on UNO that effectively introduces synthetic biology concepts to young audiences, providing a reusable and adaptable tool for science communication.
The synthetic biology-themed UNO card game we designed creatively maps biological elements to UNO mechanics:
1. Number cards represent nucleic acid bases (A, T, G, C, U) and structural components like ribose and phosphate;
2. "Skip" and "Reverse" cards correspond to DNA and RNA, respectively;
3. "Draw Two" cards symbolize codons, while "Wild Draw Four" cards represent the eight essential amino acids in humans.
4. The game also incorporates project-specific content: fruit and vegetable images on number cards, "Skip" as a kill switch, and "Reverse" as the pMA5 plasmid, "Draw Two" as lysine monomers, "Wild Draw Four" as polylysine, and "Wild" cards featuring Bacillus subtilis.
By linking gameplay with biological concepts—such as matching "base" cards to form "genetic sequences" or using "amino acid" wild cards to complete molecular "pathways"—the game offers an engaging, hands-on way to learn key synthetic biology principles, making abstract ideas accessible and memorable for players of all ages, especially for preschoolers with little knowledge reserve.
