NOTEBOOK

January-March 2025: Brainstorms and Project Design

Before the start of the project, we collaborated with the School of Life and Health Sciences at Hainan University to promote iGEM and synthetic biology across the entire university. We also publicized the recruitment for the 2025 HainanU-China team. These initiatives garnered an excellent response. Thanks to the dedicated efforts of the student leaders and the guidance of faculty members, we successfully assembled the iGEM team for the year.

Following the establishment of the team, members engaged in regular group meetings to communicate and explore solutions to societal issues that have a broad impact and can bring positive change to the world. Through extensive information research and consideration of members' interests, we turned our attention to epilepsy, a refractory disorder affecting over 50 million people globally. After initially defining the project direction, we dedicated over a month to reviewing background literature. This allowed team members to become well-versed in the fundamental aspects of epilepsy, current treatment modalities, and the numerous limitations of existing technologies. Subsequently, we held a month-long series of brainstorms. During these sessions, we explored various perspectives and investigated the potential of synthetic biology approaches to address and improve upon the existing treatment challenges. Through numerous group discussions, we ultimately finalized the overall scope of our project: using in vitro ketogenesis as an alternative to the ketogenic diet for treating epilepsy. Subsequently, we carried out in-depth clinical interviews with medical professionals, engaged in communication with stakeholders, sought advice from university professors with expertise in neurobiology and synthetic biology, and disseminated questionnaires to assess public awareness of epilepsy. Incorporating the feedback from these activities, along with targeted literature research and analysis, we held regular group meetings. Through these meetings, we continuously refined and enhanced our project design, clearly defined the research direction, and developed a project implementation framework for the upcoming six months. This framework encompasses aspects such as integrated human practices, education, and experimental timelines. By this stage, the fundamental structure of our project design had taken shape.

April-June 2025: Determination on Details of Experiments

After determining the research content of the project, it is necessary to improve and develop the established project promotion framework. It was not easy to transfer the metabolic process that generates ketone body BHB to an in vitro prokaryotic expression system. During the project development stage, we consulted the PIs of the team, who provided us with many important reference suggestions and viewpoints in the experimental design stage. Regarding the gene circuit, we reviewed numerous literature on BHB production and found various different metabolic pathways. Eventually, we focused on an existing in vitro industrial production metabolic pathway for BHB, which is simple and has been proven to be effective. However, in order to successfully express and excrete BHB in the Escherichia coli prokaryotic expression system, we added new elements encoding membrane proteins for transmembrane transport of BHB as a supplement to the original gene circuit. Through collective discussions in group meetings, we noticed that problems such as the easy formation of inclusion bodies when expressing eukaryotic proteins in the prokaryotic expression system and the specific choice of bacterial strains were expected. During this process, we simulated and reflected on the experimental plans determined at each stage, and used the work from the previous stage as a basis for learning and optimization. Eventually, we determined the complete design for the subsequent experimental implementation stage. This process was carried out and completed under the guidance of the engineering cycle of Design-Build-Test-Learn. In addition, considerations and designs related to biosafety were also completed at this stage. When team members were thinking about how to solve the problem of engineered bacteria colonization and prevent gene leakage caused by complex interactions with the intestinal flora, we creatively proposed the use of hydrogels. Through literature review and consultation with professors who have a background in materials research, our idea was proven to be completely feasible! Correspondingly, safety designs at the molecular level were also mentioned. Through the collective wisdom of team members, the idea of using siRNA-mediated gene silencing technology and suicide circuits was successfully designed, effectively ensuring the safety of our project.

July 2025: Reagent Preparation and Preliminary Experiments

In July, we completed and optimized the protocol for the project experiment to ensure its feasibility during the implementation stage. We listed the reagents and consumables that would definitely be used during the experiment and submitted the list to the PIs for them to purchase and prepare. For the experimental supplies that might be needed, we made another list and submitted it to the PIs as a supplementary plan for unexpected needs. At this stage, we conducted some preliminary experiments, such as the preparation of LB solid and liquid media, the cultivation of Escherichia coli DH5α, and the cultivation of colon cancer cells.

August-September 2025: Implementation of Experiments

In August and September, all the experimental designs were put into the laboratory for implementation. According to our experimental design, the construction of engineered bacteria expression products and the preparation of hydrogels for performance testing were carried out simultaneously, with the aim of completing a greater portion of the project's experimental content within the limited time.

Construction of Engineered Bacteria and Verification of Product Expression

August 9th: We prepared LB medium containing ampicillin, including both solid and liquid media. Considering that the plasmid backbone we used contains an ampicillin resistance gene, this medium is helpful for us to pick E. coli containing the recombinant plasmid. It is necessary to prepare these media.

August 10th: We transformed Escherichia coli DH5α into competent cells and then introduced plasmids pET-21a and pRSFDuet-1 into them for replication and amplification. The transformed Escherichia coli DH5α were then inoculated into the prepared culture medium for cultivation.

Figure 1. Map of the pET-21a Plasmid Figure 2. Map of the pRSFDuet-1 Plasmid Figure 3. Colonies of Transformed Escherichia coli DH5α

August 11th: After picking single colonies, plasmids were extracted and frozen for storage.

August 13th: We used PCR to amplify the target gene fragments of phaA, phaB, pcT, and rplO. We then used double enzyme digestion to cut the plasmids pET-21a and pRSFDuet-1 at specific sites. After that, we conducted nucleic acid electrophoresis for detection, performed gel extraction, and measured the concentration. Finally, we prepared fresh culture medium for subsequent use.

igure 4. Recombinant Plasmid pET-21a with phaA Figure 5. Recombinant Plasmid pET-21a with phaB Figure 6. Recombinant Plasmid pET-21a with pcT : Figure 7. Recombinant Plasmid pRSFDuet-1 with rplO Figure 8. Agarose Gel Electrophoresis of phaA Amplification    Figure 9. Agarose Gel Electrophoresis of phaB Amplification Figure 10. Agarose Gel Electrophoresis of pcT amplification : :  :  : Figure 11. Agarose Gel Electrophoresis of rplO amplification Figure 12. Results of Linearized Plasmid pET-21a  : : : : Figure 13. Results of Linearized Plasmid pET-21a (DNA Marker Caption) Figure 14. Results of Linearized Plasmid pRSFDuet-1      Figure 15. Results of Linearized Plasmid pRSFDuet-1 (DNA Marker Caption)

August 14th: We used seamless cloning technology to connect the target gene fragments phaA, phaB, pcT, and rplO to the specific sites of the plasmids pET-21a and pRSFDuet-1 that had been double digested. Then, we transformed Escherichia coli DH5α into competent cells and introduced the recombinant plasmids, and inoculated them into the prepared medium for cultivation.

August 15th: Unfortunately, on this day, we observed the results and found that the concentration of the recombinant plasmid containing the target gene fragment was relatively low, and the outcome was not satisfactory, making it impossible to proceed with the subsequent experiments. As a result, we were trapped in the cycle of this experimental period.

August 15th - 23rd: Repeated the above-mentioned experiment cycle.

August 24th: We sequenced the plasmid within the bacterial cells, and this process lasted approximately 10 days.

September 3rd-5th: After obtaining the sequencing results, we conducted two more days of experiments on plasmid amplification and extraction. However, we still encountered the problem of some plasmids having too low concentrations. Therefore, we had to consider changing the experimental plan, replacing the plasmids with pET-Duet-1 and pRSFDuet-1, and connecting the rplO and phaA gene fragments to pRSFDuet-1, and the phaB and pcT gene fragments to pET-Duet-1. At the same time, to avoid the expression of inclusion bodies, we also planned to add a SUMO tag to increase the solubility of the proteins encoded by the above genes.

Figure 16. Recombinant Plasmid pRSFDuet-1 with rplO&phaA Figure 17. Recombinant Plasmid pET-Duet-1 with phaB&pcT

September 6th-20th: We amplified the SUMO fragment using PCR and simultaneously repeated the seamless cloning to ligate the target gene fragment onto the corresponding plasmid backbone. After two weeks of attempts, we finally successfully constructed the recombinant plasmid with the SUMO tag and the recombinant plasmid without the SUMO tag!

Figure 18. Results of PCR Amplification of SUMO Figure 19. Results of PCR Amplification of SUMO (DNA Marker Caption)

September 21st: We transformed Escherichia coli Nissle 1917 into competent cells and divided them into two groups. We then introduced the recombinant plasmid with a SUMO tag and the recombinant plasmid without a SUMO tag into each group respectively. After that, we inoculated them onto the prepared LB medium containing glucose for cultivation.

Figure 20. Colonies of Transformed Escherichia coli Nissle 1917

September 22nd: After a period of cultivation, we collected the liquid in the liquid culture medium into a centrifuge tube. We conducted a verification experiment for the product BHB using a BHB detection kit, and the result was positive, which proved that the metabolic route we used was feasible in the prokaryotic expression system.

Figure 21. Results from Enzyme Immunoassay Analyzer

August 27th-29th: Prior to initiating the hydrogel experiment module, we first prepared the essential materials required for fabricating the hydrogel core: LB medium, 5w.t.%CaCl₂ solution, and 5w.t.% sodium alginate (SA) solution. Subsequently, E. coli was inoculated into LB medium to obtain the seed culture. We also compiled a list of materials and contacted the PIs to assist in ordering the relevant experimental supplies.

August 30th-September 1st: We proceeded to formally fabricate the hydrogel core by mixing fresh E. coli culture with sodium alginate solution and dropping the mixture into CaCl₂ solution for solidification, resulting in the formation of hydrogel beads.

Figure 22. The Inner Hydrogel Core

September 5th-7th: We began our first growth curve measurement. The prepared hydrogel cores were ground using a grinder. Experimental groups and blank control groups were designed, and each system was added to a 96-well plate. The OD600 values were measured every 4 hours using a spectrophotometer to preliminarily investigate the effects of sodium alginate and core preparation on the growth of E. coli. The results showed that sodium alginate and the preparation of the core had little effect on the growth of E. coli, and it could grow normally under encapsulation.

Figure 23. Grind the Hydrogel into Powder Using a Grinder    Figure 24. 96-Well Plate after Sample Addition

September 9th: We thawed the Caco-2 colon cancer cells stored in the laboratory and performed subculturing every 2-3 days.

September 10th-12th: We adjusted the sodium alginate solution to alkaline conditions, added dopamine hydrochloride to it, and stirred the mixture for 24 hours to allow polymerization and grafting, resulting in a PDA-SA solution. Under ice bath conditions, acrylamide and the accelerator were added and stirred until uniform, followed by the addition of the cross-linking agent and the catalyst. Ultimately, we successfully prepared a polydopamine-sodium alginate-polyacrylamide (PSP) double-network hydrogel!

September 13th: We immersed the previously prepared hydrogel cores into the PSP pre-gel solution to form a thin shell layer. These were then submerged in an MES buffer containing cross-linking agents and catalysts for 3 hours to cross-link and thereby stabilize the shell structure.

Figure 25. Hydrogel beads coated with the middle layer

September 15th-18th:Based on the results of the inner layer growth curve measurement, we narrowed the time gradient and added mid-layer treatment groups to conduct another growth curve measurement. This time, we separately ground the inner hydrogel cores and the hydrogels coated with the mid-layer, then measured the OD600 values of the resulting ground suspensions using a spectrophotometer. We found that the mid-layer exhibited a relatively low OD600 value. Therefore, using this as the baseline, we adjusted the OD600 values of each experimental group by adding their respective blank media.

Figure 26. Some experimental materials prepared for measuring the growth curve.    Figure 27. The program set for the microplate reader. Figure 28. the 96-Well Plate with our Sample Added

Subsequently, in a biosafety cabinet, we sequentially added each sample to a 96-well plate in specified proportions. Each well received 200 μL of the respective mixture, with five replicate wells per group. The groups were set up as follows:

• Group A: LB medium
• Group B: LB medium + 5% SA (mixed at a 1:1 ratio)
• Group C: LB medium + 5% SA + CaCl₂ (cross-linker) (Composition: 5 mL LB + 5 mL SA + 0.1 mL CaCl₂)
• Group D: LB medium + 5% SA + PDA solution (Composition: 5 mL LB + 4.5 mL SA + 0.5 mL PDA)
• Group E: LB medium + 5% SA + PDA solution + Initiator (Composition: 5 mL LB + 4.5 mL SA + 0.5 mL PDA + 10 μL Initiator)
• Group F: LB medium + EcN
• Group G: LB medium + 5% SA + EcN
• Group H: LB medium + 5% SA + CaCl₂ (cross-linker) + EcN
• Group I: LB medium + 5% SA + PDA + EcN
• Group J: LB medium + 5% SA + PDA solution + Initiator + EcN
• Group K: LB medium + Inner core
• Group L: LB medium + Hydrogel beads coated with the mid-layer

After the addition of samples to all wells, the OD600 values of each sample were measured continuously for 24 hours using a microplate reader. For each group, the maximum and minimum values from the five replicates were excluded, and the arithmetic mean of the remaining three values was calculated. A growth curve was plotted with time on the x-axis and the mean OD600 value on the y-axis.

Figure 29. Growth curves of different groups Figure caption: The y-axis for Δ1 represents the OD600 value obtained by subtracting Group A from Group F, representing the growth of E. coli in LB medium; the y-axis for Δ2 represents the OD600 value obtained by subtracting Group A from Group L, representing the growth of E. coli in hydrogel beads coated with the mid-layer; the y-axis for Δ3 represents the OD600 value obtained by subtracting Group E from Group J, indicating the growth of E. coli within the mid-layer material.

As illustrated in Figure 29, using the inner-layer hydrogel cores as the research subject, we designated Group A (LB) and Group B (LB + SA) as blank controls. As shown in the figure, the OD600 values of Groups A and B showed no significant changes, confirming the absence of contamination in the blank media.

A comparison between Group F (LB + EcN) and Group G (LB + SA + EcN) revealed that the addition of sodium alginate resulted in a relatively slower yet sustained increase in bacterial growth, indicating that sodium alginate has no significant toxic effect on the bacteria.Furthermore, comparing Group F (LB + EcN) with Group K (LB + inner-layer cores) demonstrated that the bacterial population within the inner-layer hydrogel cores still maintained a considerable level of growth. This result validates our hypothesis that the inner-layer hydrogel core can effectively provide nutrients to support the growth of E. coli!

Taking the hydrogel coated with the mid-layer as the research subject, we used Group A (LB) and Group E (LB + SA + PDA + Catalyst) as blank controls. However, we observed an abnormal increase in the OD600 value of Group E. Further analysis of the data revealed that Group D (LB + SA + PDA) also exhibited an abnormal rise in OD600. Since the same bottle of LB medium was used in this experiment, why did such results occur? We found ourselves facing a dilemma. Could it be that the self-polymerization and sedimentation of PDA caused its OD600 value to increase over time? After consulting relevant literature, we proposed this hypothesis. Based on this growth curve measurement experiment, we designed an additional set of experiments.

Using PDA as the research subject, we designed the following seven groups for growth curve measurement (the reagent proportions in each system remained consistent with the previous experiment):

1. PDA solution
2. PDA + 5% SA
3. PDA + 5% SA + Initiator
4. PDA (sterilized with 75% ethanol)
5. PDA (sterilized with 75% ethanol) + LB medium
6. PDA (sterilized with 75% ethanol) + 5% SA + LB medium
7. PDA (sterilized with 75% ethanol) + 5% SA + LB medium + Initiator

We observed that the OD600 value of the PDA solution sterilized with ethanol (Group ④) actually decreased to some extent over time. Literature review suggests that polydopamine nanoparticles form a colloidal suspension in solution, which gradually settles toward the bottom of the well due to gravity, leading to a reduction in the measured apparent OD value. Furthermore, according to the growth curves, we found that the other groups containing PDA solution (Groups ⑤, ⑥, and ⑦) all exhibited varying degrees of increase in OD600. This may be attributed to physical adsorption and electrostatic complexation between PDA and either LB medium or sodium alginate, resulting in the formation of larger, more complex aggregates or complexes with enhanced light-scattering capabilities.

Therefore, we utilized the aforementioned supplementary experiments to revise and further process the original data. By applying the difference method, with the OD600 values resulting from E. coli in different media as the vertical axis and time as the horizontal axis, we plotted additional Group 4. The results indicate that E. coli in both the intermediate layer material and the hydrogel beads coated with the intermediate layer exhibited stable growth at a certain rate, demonstrating that the intermediate layer material and coating method had no significant impact on the growth of E. coli.

September 19th-21th: We prepared the hydrogel cores using the same method and subsequently coated them with the mid-layer in preparation for the application of the outer-layer coating. Meanwhile, we performed cell passaging in preparation for verifying the adhesive properties of the middle layer coating.

Figure 30. Subculture of Colorectal Cancer Cells Caco-2 Figure 31. Subculture of Colorectal Cancer Cells Caco-2

September 23rd: We used passaged Caco-2 cells to verify the adhesiveness of the middle-layer coating. Hydrogel beads encapsulated with the middle layer were placed into cell culture flasks, allowing them to come into contact with adherent cells. The position of each bead in the culture flask was recorded by photography. Subsequently, the culture flasks were inverted and placed on a shaker, which was operated at a specific rotational speed. Under different rotational speeds, photographs were taken to record the status of the beads in the flasks, and the number of detached beads was counted to evaluate the adhesiveness of the middle-layer PSP hydrogel.

Figure 32. the Detachment of Hydrogel Beads at 10-Minute Intervals within the 0–50 Minute Period. Figure 33. Hydrogel Bead Detachment Curve

We recorded the number of detached hydrogel beads in the groups with and without the middle-layer material encapsulation at different time points. The average value was calculated from two replicate groups, and line charts and box plots were plotted. The results showed that over time, no hydrogel beads detached from the group encapsulated with the middle-layer material, while the group without the middle-layer material encapsulation exhibited varying degrees of bead detachment at each time point. This indicates that the middle-layer PSP hydrogel has strong adhesiveness.

We also conducted a supplementary experiment using empty flasks without cells as a control. It was found that the detachment rate of both the inner-layer and middle-layer coatings increased significantly, which suggests that the cells simulating the intestinal environment play a crucial role.

September24th:We prepared the materials required for fabricating the outer layer of the hydrogel, such as: 1w.t.% sodium alginate solution, 3w.t.% CaCl₂ solution, and phosphate-buffered saline (PBS).

September 25th: We employed a freezing-based reverse spherification method to apply the outer coating. First, the hydrogel beads with the completed mid-layer were frozen for 5 hours, then rapidly immersed in a 3% CaCl₂ solution and stirred at a low speed for 20 minutes to allow cross-linking. After rinsing, they were soaked in a 1% sodium alginate solution for 15 minutes, resulting in the formation of a calcium alginate gel membrane.

Figure 34. the Middle-Layer-Encapsulated Hydrogel after 5 Hours of Freezing    Figure 35. Hydrogel Beads Coated with a Sodium Alginate Gel Film

September 24th-25th: We proceeded to verify the pH responsiveness of the outer calcium alginate gel membrane. The intact three-layer hydrogel was placed in PBS solutions of varying pH levels (1.29, 3.64, 5.7, 7.06, 9.77, 11.1). It was removed and weighed every 10 minutes to calculate the swelling ratio. Swelling ratio-time curves were plotted to analyze its pH responsiveness.

Figure 36. Experimental materials prepared for verifying the pH responsiveness of the outer calcium alginate gel membrane. Figure 37. It can be observed that the hydrogel beads already exhibit different textures under different pH conditions. Figure 38. The State of the Hydrogel at pH 1.29 after 50 Minutes  Figure 39. The state of the hydrogel at pH 7.06 after 50 minutes  Figure 40. The state of the hydrogel at pH 9.77 after 50 minutes Figure 41. Changes in the Mass and Swelling Ratio of the Hydrogel under Different pH Conditions.

As shown in Figures 38, 39, and 40, at the 50-minute mark, under acidic conditions with a pH of 1.29 (simulating the gastric environment), the hydrogel beads were smaller than their initial size. At a pH of 7.06 (simulating the weakly alkaline intestinal environment), the outer layer of the hydrogel beads swelled but did not burst; however, when the pH reached 9.77, some of the hydrogel beads burst.

As shown in the figure 41, it can be observed that under acidic conditions (pH=1.29 simulating gastric fluid), the mass of the hydrogel gradually decreases over time, accompanied by a reduction in the swelling ratio, indicating contraction of the outer calcium alginate film. Under neutral conditions (pH=7.06), the mass of the hydrogel gradually increases over time, along with a rise in the swelling ratio, demonstrating swelling of the outer calcium alginate film. Under alkaline conditions (pH=11.1), both the mass and swelling ratio of the hydrogel drop sharply over time, suggesting eventual dissolution of the outer calcium alginate film, making weight measurement impossible.

The experimental results are in perfect agreement with our hypothesis! They confirm that the outer calcium alginate film exhibits the expected pH responsiveness: in the low-pH gastric fluid, the outer layer contracts to form a dense, insoluble membrane, which can effectively prevent gastric acid and proteases from damaging the internally encapsulated substances while avoiding their premature release; in the nearly neutral-pH intestinal tract, the outer network becomes loose, with increased permeability and adhesiveness, allowing it to successfully colonize the intestinal tract, and enabling the internally encapsulated substances to diffuse and release, further facilitating the synthesis and excretion of BHB!!

Over the past year, our team has held numerous group meetings. From the first gathering after new member recruitment to joint brainstorming sessions, idea exchanges, roadshow preparations, and progress updates—each meeting marked a milestone in the project's advancement. Every idea proposed by team members added a fresh, brilliant dimension to our project.

Behind these meetings lies the warm support from every member of HainanU-China. Driven by their passion for iGEM and a strong spirit of teamwork, everyone supported one another. Through countless discussions, we collectively developed our project "Epilepsy Shield".

These are the records of some of our group meetings; some meetings were not documented.