iGEM 2025 — Team hainanu-china

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# NOTEBOOK

From Design to Experiment
1

January-March
2025: Brainstorms and Project Design
April-June
2025: Determination on Details of Experiments
July
2025: Reagent Preparation and Preliminary Experiments
August-September
2025: Implementation of Experiments

Experiments on BHB Synthesis
2

Construction
of Engineered Bacteria and Verification of Product Expression

Experiments on Hydrogels
3

Group Meeting Minutes
4

From Design to Experiment

**## 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.

Experiments on BHB Synthesis

**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.

![](https://static.igem.wiki/teams/5911/notebook/note1.webp)

![](https://static.igem.wiki/teams/5911/notebook/note2.webp)

Figure 1. Map of the pET-21a Plasmid Figure 2. Map of the pRSFDuet-1
Plasmid

![](https://static.igem.wiki/teams/5911/notebook/note3.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/note4.webp)

![](https://static.igem.wiki/teams/5911/notebook/note5.webp)

igure 4. Recombinant Plasmid pET-21a with phaA Figure 5. Recombinant
Plasmid pET-21a with phaB

![](https://static.igem.wiki/teams/5911/notebook/note6.webp)

![](https://static.igem.wiki/teams/5911/notebook/note7.webp)

Figure 6. Recombinant Plasmid pET-21a with pcT : Figure 7.
Recombinant Plasmid pRSFDuet-1 with rplO

![](https://static.igem.wiki/teams/5911/notebook/note8.webp)

![](https://static.igem.wiki/teams/5911/notebook/note9.webp)

Figure 8. Agarose Gel Electrophoresis of phaA
Amplification    Figure 9. Agarose Gel Electrophoresis of phaB
Amplification

![](https://static.igem.wiki/teams/5911/notebook/note10.webp)

![](https://static.igem.wiki/teams/5911/notebook/note11.webp)

Figure 10. Agarose Gel Electrophoresis of pcT amplification : :
 :  : Figure 11. Agarose Gel Electrophoresis of rplO amplification

![](https://static.igem.wiki/teams/5911/notebook/note12.webp)

![](https://static.igem.wiki/teams/5911/notebook/note13.webp)

Figure 12. Results of Linearized Plasmid pET-21a
 : : : : Figure 13. Results of Linearized Plasmid pET-21a (DNA Marker
Caption)

![](https://static.igem.wiki/teams/5911/notebook/note14.webp)

![](https://static.igem.wiki/teams/5911/notebook/note15.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/note16.webp)

Figure 16. Recombinant Plasmid pRSFDuet-1 with rplO&phaA

![](https://static.igem.wiki/teams/5911/notebook/note17.webp)

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!

![](https://static.igem.wiki/teams/5911/notebook/note18.webp)

Figure 18. Results of PCR Amplification of SUMO

![](https://static.igem.wiki/teams/5911/notebook/note19.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/note20.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/note21.webp)

Figure 21. Results from Enzyme Immunoassay Analyzer

Experiments on Hydrogels

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.

![](https://static.igem.wiki/teams/5911/notebook/22.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/23.webp)

![](https://static.igem.wiki/teams/5911/notebook/24.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/25.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/26.webp)

![](https://static.igem.wiki/teams/5911/notebook/27.webp)

Figure 26. Some experimental materials prepared for measuring the
growth curve.    Figure 27. The program set for the microplate
reader.

![](https://static.igem.wiki/teams/5911/notebook/28-96.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/29.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/30-1.webp)

![](https://static.igem.wiki/teams/5911/notebook/30-2.webp)

Figure 30. Subculture of Colorectal Cancer Cells Caco-2

![](https://static.igem.wiki/teams/5911/notebook/31.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/0.webp)

![](https://static.igem.wiki/teams/5911/notebook/1.webp)

![](https://static.igem.wiki/teams/5911/notebook/2.webp)

![](https://static.igem.wiki/teams/5911/notebook/3.webp)

![](https://static.igem.wiki/teams/5911/notebook/4.webp)

![](https://static.igem.wiki/teams/5911/notebook/5.webp)

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

![](https://static.igem.wiki/teams/5911/notebook/33.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/34.webp)

![](https://static.igem.wiki/teams/5911/notebook/35.webp)

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.

![](https://static.igem.wiki/teams/5911/notebook/36.webp)

Figure 36. Experimental materials prepared for verifying the pH
responsiveness of the outer calcium alginate gel membrane.

![](https://static.igem.wiki/teams/5911/notebook/37.webp)

Figure 37. It can be observed that the hydrogel beads already exhibit
different textures under different pH conditions.

![](https://static.igem.wiki/teams/5911/notebook/38.webp)

![](https://static.igem.wiki/teams/5911/notebook/39.webp)

![](https://static.igem.wiki/teams/5911/notebook/40.webp)

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

![](https://static.igem.wiki/teams/5911/notebook/41.webp)

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!!

Group Meeting Minutes

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.

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# ENGINEERING

EEG QuickLab: From Hardware Realities to a
Lightweight Educational EEG Platform
C1

Selection of the microbiota regulation module
scheme
C2

Construction of recombinant plasmids
C3

Design, Preparation and Characterization of
Bacteria-Encapsulated Hydrogels
C4

Evaluation of in Vitro Basic Gene Leakage of
Engineered Bacteria-Hydrogel System.
C5

References
6

Cycle 1: EEG-based epilepsy monitoring and prediction model

**## Design**

In the course of clinical treatment, the EEG signals of epilepsy patients during seizures
often contain characteristic spike waves, slow waves, and spike-and-slow wave complexes.
These features can be utilized to evaluate the patients' health conditions and seizure
risks. However, in practical clinical settings, the judgment usually relies on the
extensive experience of clinicians. This not only imposes a significant burden on
doctors but also introduces uncertainties for patients. To provide a reliable adjunct
for clinical treatment, we are dedicated to developing an EEG-based epilepsy monitoring
and prediction model. The epilepsy dataset we utilized was sourced from
OpenNEURO(https://openneuro.org/datasets/ds003555/versions/1.0.1), which contains
electroencephalogram (EEG) recordings of high-frequency oscillations (HFO) from 30
pediatric epilepsy patients. The authors also shared code on GitHub detailing data
processing workflows including signal filtering, baseline calibration, and event
detection. By analyzing EEG data from these patients and applying the provided GitHub
code for feature computation, we generated labeled time segments of epileptic seizures.
However, the labels contained false positives mixed with true positives. To further
optimize true positive prediction, we implemented additional features beyond those
specified by the authors, including L1 norm calculation, Shannon entropy analysis,
sample entropy metrics, and permutation entropy evaluation. Through automated
identification of these anomalous feature values, we conducted predictive classification
tasks to distinguish between true and false positives using deep learning models.

**## Build**

In this section, we extracted time-domain and frequency-domain features from columns 6 to
14 of the TSV file for machine learning integration. Using column 15's true
positive/false positive predictions as the reference target, we developed three models:
a random forest model, a CNN model, and an LSTM-CNN hybrid model. After evaluating
performance metrics including accuracy rates across these models, we ultimately selected
the optimized CNN model as the final implementation.

![](https://static.igem.wiki/teams/5911/engineer/1-1.avif)

![](https://static.igem.wiki/teams/5911/engineer/1-2.avif)

Figure 1. Partial content of the dataset

![](https://static.igem.wiki/teams/5911/engineer/2.avif)

Figure 2. Analysis and Characteristics of Various Waveforms in the
Fp1-Fp2 Channel
**## Test**

During the model's testing phase, we use the accuracy result of model training as the
measurement standard. We divide the existing dataset into a training set and a
validation set. We input the positive and negative sets of the training set into the
model for learning. After completion, we input the validation set to check the training
results. The epilepsy detection model was evaluated on the independent test set with an
accuracy of 80.85% and a sensitivity of 82.3%. The prediction confidence of a single
prediction was sometimes low, indicating the uncertainty of decision.

![](https://static.igem.wiki/teams/5911/engineer/3.avif)

Figure 3. Model Training Results
**## Learn**

Analysis shows that data imbalance and the loss of time-domain information due to
frequency-domain features lead to performance limitations. More data needs to be
collected, time-domain features should be integrated, and the model architecture should
be optimized, such as adding LSTM to enhance generalization ability. By combining
machine learning methods, the accuracy of distinguishing the resting-state EEG of normal
patients from that of epilepsy patients can reach over 90%, but the model for
calculating the true positive/false positive labels during epileptic seizures needs to
be further optimized based on specific criteria. In the next cycle of the DBTL
engineering loop, we plan to divide the dataset more reasonably to enhance the model's
sensitivity, accuracy and precision.

Cycle 2: Selection of the microbiota regulation module scheme

**## Design**

Our design goal is to construct an efficient, safe, stable and highly compatible BHB
synthesis pathway in engineered E.coli Nissle 1917 ( EcN ). We aim to design a
self-sustaining metabolic cycle. The innovation lies in the use of the two-way reaction
characteristics of PCT : the CoA group on BHB-CoA is transferred to acetic acid to form
the final product BHB, and acetic acid is converted to acetyl-CoA, which is the starting
substrate of the BHB synthesis pathway. This design realizes the regeneration of
acetyl-CoA, bringing multiple theoretical advantages including reducing metabolic load,
improving pathway sustainability, and solving the accumulation toxicity of potential
metabolites.

The intestine is rich in short-chain fatty acids ( SCFAs ), of which acetic acid is one
of the most important components. The design of the PCT pathway cleverly transforms this
environmental feature into an advantage. Our engineered bacteria can directly use
intestinal endogenous acetic acid as a ' raw material ' to drive the regeneration of
acetyl-CoA, reducing the competition for host direct energy substances ( such as glucose
) and reducing the risk of potential interference with host metabolism. This pathway
enables engineered bacteria to be more harmoniously integrated into the intestinal
micro-ecosystem, and its metabolic activity complements the intestinal chemical
environment, improving the stability of colonization and the persistence of therapeutic
effects.
Intestinal short-chain fatty acids ( SCFAs ), of which acetic acid is one of the most
important components. The design of the PCT pathway cleverly transforms this
environmental feature into an advantage. Our engineered bacteria can directly use
intestinal endogenous acetic acid as a ' raw material ' to drive the regeneration of
acetyl-CoA, reducing the competition for host direct energy substances ( such as glucose
) and reducing the risk of potential interference with host metabolism. This pathway
enables engineered bacteria to be more harmoniously integrated into the intestinal
micro-ecosystem, and its metabolic activity complements the intestinal chemical
environment, improving the stability of colonization and the persistence of therapeutic
effects.

![](https://static.igem.wiki/teams/5911/engineer/4.avif)

Figure 4. Three Different BHB Synthesis Pathways

Before determining the final scheme, we systematically evaluated other major BHB
synthesis pathways reported in the literature and weighed them based on our design goals
:

**PhaZ pathway ( polymerization-depolymerization pathway ) :**

Mechanism : Polyhydroxybutyrate ( P ( 3HB ) ) particles were synthesized by PhaA, PhaB,
and PhaC, and then BHB was produced by PhaZ depolymerization.
Disadvantages : This approach is too lengthy and needs to coordinate the two processes
of aggregation and disaggregation that may conflict in time and space ; due to its long
cycle of more than 100 hours and low productivity ( ~ 0.07 g / L / h ), it is completely
unable to meet the requirements of rapid response and dynamic regulation required for in
vivo treatment.

**TesB pathway ( thioesterase pathway ) :**

Mechanism : BHB-CoA was synthesized by PhaA and PhaB, and then hydrolyzed by
thioesterase ( TesB ) to produce BHB.

Disadvantages : Although simpler than the PhaZ pathway, the TesB-catalyzed hydrolysis
reaction is a one-way, irreversible consumption process. The production of each BHB
molecule permanently consumes one acetyl-CoA and one CoA, which will impose an
increasing burden on the central metabolism and lack sustainability, which may affect
the long-term survival and function of the engineered bacteria.

**Ptb-Buk pathway ( phosphotransferase pathway ) :**

Mechanism : BHB-CoA is converted to BHB by phosphotransbutyrase ( Ptb ) and butyrate
kinase ( Buk ).

Disadvantages : Similar to the TesB pathway, it is also a CoA consumption pathway, and
requires ATP consumption, and does not have an advantage in energy economics.

After comparison, the PCT approach has become the best choice to meet all our core design
standards due to its outstanding theoretical advantages of regeneration cycle,
environmental compatibility, and high efficiency and sustainability.

**## Build**

We assigned specific functions and optimization considerations to each core enzyme
element :
PhaA ( β-ketothiolase, derived from Ralstonia eutropha ) catalyzes the Claisen
condensation reaction of two molecules of acetyl-CoA to produce acetyl-CoA. This is the
rate-limiting initial step of the entire pathway. In order to ensure high catalytic
efficiency, we selected the PhaA isoenzyme, which has been characterized in the bacteria
and has high affinity for acetyl-CoA. At the same time, codon optimization was performed
on its coding sequence to adapt to the translation system of E.coli and avoid low
expression efficiency and translation errors caused by rare codons.

PhaB ( acetylacetyl-CoA reductase, derived from Ralstonia eutropha ) uses NADPH as a
coenzyme to stereoselectively reduce acetylacetyl-CoA to ( R ) -BHB-CoA. This is a key
step in determining the chiral purity of the product. The PhaB variant with high ( R )
-stereoselectivity was strictly selected to ensure the optical purity of the product
more than 99 % from the source. At the same time, we focused on its coenzyme circulation
efficiency with NADPH / NADP + to ensure that it can be coordinated with the reducing
power supply of host cells.

**## Test**

Before starting the wet experiment, we conducted a systematic comparative analysis of the
public performance data of multiple channels, which constitutes a crucial ' test ' link
in our decision-making process. We have established an assessment framework that focuses
on titer , productivity and pathway sustainability.

Existing studies have shown that although the PhaZ pathway can obtain a higher final
yield ( such as 7.3 g / L ), it requires a two-step fermentation process of more than
100 hours, resulting in a generally low volumetric productivity ( about 0.07 g / L / h
). For in vivo treatment that requires rapid response and dynamic regulation, a lengthy
production cycle is unacceptable. Therefore, despite its high absolute yield, low
productivity makes it unsuitable for our application scenarios.
Liu et al. ( 2007 ) reported that the TesB pathway can achieve 4.0 g / L production
within 24-48 hours, and the productivity is 0.17 g / L / h. The TesB pathway is more
direct, but its hydrolysis consumes coenzyme A ( CoA ) and cannot regenerate acetyl-CoA.
This means that the synthesis of each BHB molecule is at the expense of permanent
consumption of central metabolites, which may limit the sustainability of long-term
production.

The direct study of Matsumoto et al. ( 2013 ) under the same strain background and
comparable conditions provided us with key evidence. The data showed that the yield of
the PCT pathway was as high as 5.2 g / L and the productivity reached 0.22 g / L / h
after acetic acid supplementation, which was significantly higher than that of the PhaZ
pathway ( 0.07 g / L / h ) and the TesB pathway ( 0.17 g / L / h ), which proved its
advantage in speed.

In terms of mechanism verification, the literature confirmed by 13C isotope labeling
experiments that exogenous acetic acid was directly integrated into BHB molecules,
providing solid experimental evidence for the ' acetyl-CoA regeneration ' mechanism,
which is far better than pure theoretical speculation. In terms of product quality, the
purity of ( R ) -BHB enantiomers reported in the literature was as high as 99.2 %, which
met the treatment requirements. In terms of environmental adaptability, the yield
increases with the increase of acetic acid concentration, which is perfectly consistent
with the acetic acid-rich human intestinal environment, indicating that our engineered
bacteria can effectively utilize endogenous acetic acid in the body to achieve more
stable and sustainable production.

**## Learn**

Through systematic comparison and analysis of these key literature data, we have obtained
clear and reliable conclusions :

The PCT pathway performs best in key performance indicators ( productivity ) and can
meet the needs of in vivo treatment for rapid onset.

Its unique ' acetyl-CoA regeneration ' mechanism has been strongly supported by
experimental data, laying a metabolic foundation for long-term and sustainable
production.

This pathway has a high degree of internal compatibility with the target colonization
environment ( gut ), which is a ' born for the gut ' pathway.

Therefore, this in-depth test based on literature data convinces us that choosing the
PhaA-PhaB-PCT pathway is a data-driven and rational optimal decision. It not only
surpasses the alternative in performance, but also its inherent characteristics are
highly consistent with our final application goals, laying a solid foundation for
subsequent gene circuit design and security control. This method minimizes the risk of R
& D and embodies the core spirit of rational engineering design of synthetic biology.

Cycle 3: Construction of recombinant plasmids

**## Design**

Centering on the core objective of achieving BHB synthesis in Escherichia coli Nissle
1917, based on the previously screened BHB synthesis pathway, it was determined that
four key target gene fragments, namely phaA, phaB, pcT, and rplO, need to be introduced.
After collaborative discussion with the PI on the plasmid construction strategy, the
initial design was to clone phaA, phaB, and pcT genes respectively using the pET-21a
vector (constructing three recombinant plasmids), while cloning the rplO gene using the
pRSFDuet-1 vector (constructing one recombinant plasmid). Ultimately, all genes would be
introduced into the host bacteria through "co-transformation of four plasmids". The core
consideration of this design is the "single gene - single vector" modular construction,
which has the advantages of intuitive operation and clear steps, and initially meets the
basic requirements for pathway assembly.

![](https://static.igem.wiki/teams/5911/engineer/5.avif)

![](https://static.igem.wiki/teams/5911/engineer/6.avif)

Figure 5. Map of the pET-21a Plasmid    Figure 6.
Map of the pRSFDuet-1 Plasmid

![](https://static.igem.wiki/teams/5911/engineer/7.avif)

![](https://static.igem.wiki/teams/5911/engineer/8.avif)

Figure 7. Recombinant Plasmid pET-21a with
phaA    Figure 8. Recombinant Plasmid pET-21a with phaB

![](https://static.igem.wiki/teams/5911/engineer/9.avif)

![](https://static.igem.wiki/teams/5911/engineer/10.avif)

Figure 9. Recombinant Pasmid pET-21a with
pcT    Figure 10. Recombinant Plasmid pRSFDuet-1 with rplO
**## Build**

Strictly follow the design plan to carry out the experiment:

①Firstly, specifically amplify the four target gene fragments of phaA, phaB, pcT, and
rplO through PCR technology to ensure the length and sequence accuracy of the target
fragments;

②Use seamless cloning technology to connect each gene to the corresponding vector
(phaA/phaB/pcT to pET-21a, rplO to pRSFDuet-1), completing the construction of four
recombinant plasmids;

③To achieve efficient amplification of the plasmids, first transfer the recombinant
plasmids into the competent cells of Escherichia coli DH5α (DH5α is a plasmid
replication-optimized strain that can quickly obtain high-copy plasmids), and plan to
extract the plasmids to obtain high-concentration products, and then transfer them to
the final host Escherichia coli Nissle 1917 to complete the initial construction of the
engineered bacteria.

**## Test**

Centering on "verifying the feasibility of the design plan", two major test indicators
were set:

①Whether the concentration of the plasmid after amplification meets the subsequent
transformation requirements;

②The host adaptability of the co-transformation of four plasmids.

During the experimental verification, key problems were discovered:

①The plasmid extraction experiment was repeated four times, and the concentration of the
recombinant plasmid obtained each time was extremely low, far from reaching the
concentration threshold required for the transformation of engineered bacteria, directly
leading to the inability to proceed with the subsequent transformation experiments;

②Combined with the discussion of the PI team and further analysis: when four plasmids are
simultaneously introduced into a single host bacterium, it will significantly increase
the burden of exogenous gene expression on the bacteria (including promoter competition,
consumption of plasmid replication resources, etc.), and both theoretically and
practically, it has been confirmed that there are inherent defects in the success rate
of this plan.

In conclusion, the test results clearly show that the initial design plan did not meet
the expectations in both "plasmid yield" and "host compatibility", and targeted
optimization is needed.

**## Learn**

In response to the issue of "low concentration and high host stress due to excessive
plasmid quantity" identified during the Test phase, a scheme optimization was carried
out:

①Iteration of vector selection: Switch to dual-promoter vectors pET-Duet-1 and pRSFDuet-1
(both support independent expression of two genes on the same vector), and clone phaB
and pcT in tandem to pET-Duet-1, and phaA and rplO in tandem to pRSFDuet-1, constructing
only two recombinant plasmids;

②Optimization logic: Through the integration of "multiple genes - single vector", not
only the number of plasmid constructions is reduced (from 4 to 2), simplifying the
operation complexity and reducing the risk of extraction failure, but also the exogenous
expression pressure on the host bacteria is significantly alleviated, fundamentally
solving the compatibility issue of co-transformation.

Ultimately, through experimental verification, the optimized dual-plasmid
co-transformation scheme successfully achieved the construction of engineered bacteria,
and the BHB synthesis pathway could be normally initiated, confirming the feasibility of
this iterative design and completing the closed-loop optimization of the DBTL cycle.

![](https://static.igem.wiki/teams/5911/engineer/11.avif)

![](https://static.igem.wiki/teams/5911/engineer/12.avif)

Figure 11. Recombinant Plasmid pRSFDuet-1 with
rplO&phaA    Figure 12. Recombinant Plasmid pET-Duet-1 with phaB&pcT

Cycle 4：Design, Preparation and Characterization of Bacteria-Encapsulated
Hydrogels

**## Design**

We envision using hydrogels to encapsulate engineered bacteria, which can physically
adhere directly to the intestinal tract without the need to introduce additional
elements into the bacteria for expressing adhesive substances. This design can
physically isolate the engineered bacteria from the intestinal flora to avoid horizontal
exchange of genetic material and ensure the stable presence of the bacteria in the
digestive tract.

Through literature review, we have designed a hydrogel mainly composed of sodium alginate
(SA) and polydopamine (PDA), following a three-layer structure concept. It consists of
an outer pH-responsive layer, a middle adhesive layer, and an inner nutrient layer. The
outermost layer is a hydrogel made of alginate, which features responsiveness to the
weakly alkaline pH of the intestinal tract and easily swells and ruptures in the
intestine to expose the middle layer. The middle layer is a coating composed of
polydopamine, sodium alginate, and polyacrylamide, which has adhesiveness to adhere to
the intestinal tract for colonization. Additionally, the pore size of this middle layer
does not allow macromolecules such as DNA to pass through, thereby preventing gene
leakage. The innermost layer is a mixture of alginate and E. coli culture medium, which
provides nutrients for the growth of E. coli.

![](https://static.igem.wiki/teams/5911/engineer/13-1.avif)

![](https://static.igem.wiki/teams/5911/engineer/13-2.avif)

![](https://static.igem.wiki/teams/5911/engineer/13-3.avif)

![](https://static.igem.wiki/teams/5911/engineer/13-4.avif)

Figure 13. Schematic Diagram of the Three-Layer Smart Hydrogel System
**## Biuld**

1.Preparation of Hydrogel Alginate Core

Mix fresh bacterial culture (10⁸–10⁹ cells/mL in LB medium) with a 5 w.t.% sodium
alginate (SA) solution at a 1:1 volume ratio to achieve a final SA concentration of 2.5
w.t.%. Load this bacteria-alginate premix into a syringe, then drop it into a
pre-sterilized 5 w.t.% CaCl₂ solution. Allow solidification for 15 minutes to form
bead-like droplets with a controllable diameter of approximately 3 mm.

2.Preparation of Hydrogel Adhesive Middle Layer

Add a certain amount of dopamine hydrochloride (DA) to a weakly alkaline 2 wt.% sodium
alginate (SA) solution, and stir at room temperature for 24 hours to obtain a PDA-SA
solution.

Under an ice-water bath (approximately 4℃), sequentially add a certain amount of
acrylamide (AM) and cross-linking agent to the above solution. After thorough stirring,
add initiator and catalyst to the mixed solution. Mix well, transfer the mixture into a
glass petri dish, and immerse the alginate cores into the pre-gel solution to form a
middle-layer coating. Immerse the hydrogels in MES buffer (0.1 M MES, 0.5 M NaCl, pH
6.0) containing cross-linking agent and catalyst to form covalent bonds between the
alginate and polyacrylamide networks, and maintain this state for 3 hours.

3.Preparation of Hydrogel pH-Responsive Outer Layer

Place the hydrogel beads with the middle layer prepared into a -20 °C refrigerator for
freezing for 5 hours. Then immerse them into a solution containing 3% calcium ions and
1% SA. The calcium ions on the surface of the beads react with SA to form a calcium
alginate gel film. Store the final hydrogels in a humid environment.

**## Test**

Conduct experiments for safety evaluation, outer-layer pH responsiveness verification,
and middle-layer adhesion performance verification

By determining the growth curve of E. coli in the hydrogel beads, we verified that the
designed hydrogel does not affect the normal growth of engineered bacteria, indicating
that the material is relatively safe for bacteria. Then, we used passaged colon cancer
cells to verify the adhesion performance of the hydrogels coated with the middle layer.
Finally, by measuring the swelling ratio of the prepared hydrogel beads in PBS buffers
with different pH values, we verified their responsiveness to the weakly alkaline pH of
the intestinal tract.

**1.Safety Evaluation – Determination of E. coli Growth Curve**

Place hydrogel samples coated with the middle layer and samples with only the inner core
into centrifuge tubes containing 1 mL of liquid LB medium, respectively. Homogenize
using 3.2 mm stainless steel beads at a frequency of 60 Hz for 20 minutes on a tissue
grinder. To quantify cell density, measure the homogenized samples with a
spectrophotometer and adjust their concentrations appropriately. Using this as a
reference, adjust the absorbance values of each experimental group, record the ratios,
and add each sample to a 96-well plate in proportion. Measure the absorbance values of
each sample continuously for 24 hours using a microplate reader, and plot the growth
curves. The groups are divided as follows:

* Group A: LB medium
* Group B: LB medium + 5% SA (1:1 mixture)
* Group C: LB medium + 5% SA + CaCl₂ (cross-linking agent)
* Group D: LB medium + 5% SA + PDA solution (system: 5 mL LB + 4.5 mL SA + 0.5 mL
  PDA)
* Group E: LB medium + 5% SA + PDA solution + initiator (system: 5 mL LB + 4.5 mL
  SA + 0.5 mL PDA + 10 μL initiator)
* Group F: LB medium + E. coli (EcN)
* Group G: LB medium + 5% SA + E. coli (EcN)
* Group H: LB medium + 5% SA + CaCl₂ (cross-linking agent) + E. coli (EcN)
* Group I: LB medium + 5% SA + PDA + E. coli (EcN)
* Group J: LB medium + 5% SA + PDA solution + initiator + E. coli (EcN)
* Group K: LB medium + inner core
* Group L: LB medium + hydrogel beads after coating with middle layer

Add 200 μL of each above system to each well of the 96-well plate, with 5 wells per
group. After excluding the maximum and minimum values, calculate the arithmetic mean.
Plot the growth curve with time as the abscissa and OD₆₀₀ as the ordinate.

The result validates our hypothesis that the inner-layer hydrogel core can effectively
provide nutrients to support the growth of E. coli!

However, when the middle layer was used as a control, issues arose with the experimental
data. We have analyzed and discussed these issues, and further details can be found in
the "Learn" section.

![](https://static.igem.wiki/teams/5911/engineer/14.avif)

Figure 14. Growth Curves of Different Groups

**2. Verification of the Adhesion Performance of the Middle Layer**

Take out 4 bottles of passaged Caco-2 cells from the incubator and divide them equally
into two groups. Inside a biosafety cabinet, aspirate and discard the medium. Place 100
hydrogel cores and 100 hydrogel beads coated with the middle layer into the culture
bottles respectively, ensuring the beads are evenly dispersed. Invert the culture
bottles containing the beads on a shaker, and take photos to record the position of each
bead in the culture bottle. Shake the shaker at a certain rotational speed, take photos
every 5 minutes to observe and record the changes in the position of the beads in the
culture bottle, and continue this process for 50 minutes. Repeat the above experiment
with the shaker rotating at different speeds, and record the changes in the position of
the beads under different rotational speeds.

The results showed that over time, no hydrogel beads detached in the group coated with
the middle layer material, whereas the uncoated group exhibited varying degrees of
detachment at each time point. This indicates that the middle PSP hydrogel layer
possesses strong adhesive properties.

![](https://static.igem.wiki/teams/5911/engineer/15.avif)

Figure 15. Hydrogel Bead Detachment Curve

**3. Verification of the pH Responsiveness of the Outer Layer**

Place the prepared hydrogels at room temperature for 3 hours, then weigh them to obtain
the initial weight (W₀). Immerse the samples separately into beakers containing
phosphate-buffered saline (PBS) with pH values of 1.5, 7.4, and 11.0. Every 10 minutes,
take out the samples, quickly blot the moisture on the sample surface with filter paper,
and weigh them, recording the weight as Wₛ. This measurement process is conducted in a
constant temperature and humidity chamber, with the temperature set at 37°C and the
humidity at 60%. The swelling ratio is calculated using the formula: Swelling Ratio =
(Wₛ - W₀) / W₀ × 100%

The data shows in the near-neutral intestinal environment, the outer network becomes
loose with increased permeability, allowing the diffusion and release of the
encapsulated substances.

![](https://static.igem.wiki/teams/5911/engineer/16.avif)

Figure 16. Changes in the Mass and Swelling Ratio of the Hydrogel under
Different pH Conditions.
**## Learn**

1. How to enhance the adhesiveness of hydrogels has always been a challenging problem for
us. After reviewing numerous literatures, we finally modified the middle-layer hydrogel
material by adding dopamine hydrochloride, which was grafted onto sodium alginate
through Michael addition reaction and Schiff base reaction, greatly enhancing the
adhesiveness of the material.

2. When coating the middle layer of the hydrogel, in the initial experiments, the
prepared hydrogel was too brittle and fragile to meet our expected results. After
literature research and reflection, we found that it was because the hydrogel core was
placed in the PDA-SA solution for too long, leading to excessive cross-linking and
reduced mechanical properties. In the second preparation, we reduced the time of placing
the hydrogel core in the PDA-SA solution, and obtained hydrogels with good mechanical
properties.

3. When passaging Caco-2 cells, cell clumping is prone to occur. The reason may be that
after adding serum-containing complete medium to terminate digestion, sufficient
pipetting is not performed immediately, and the remaining trypsin will continue to act,
causing cells to aggregate more easily due to damage. We found that pipetting the cell
clumps repeatedly with appropriate force, especially along the bottom and side walls of
the culture flask, can alleviate this problem and enable the cells to grow better.

4. Taking the hydrogel encapsulated with the middle layer as the research object, we
analyzed the data and found anomalies. We hypothesized that the self-aggregation of PDA
(polydopamine) caused its OD600 value to increase over time. Therefore, we took PDA as
the research object and further determined the growth curve. Using the difference
method, we obtained the OD600 values contributed by Escherichia coli (E. coli) in
different media.

The results showed that E. coli could grow stably at a certain growth rate both in the
middle-layer material and the hydrogel beads encapsulated with the middle layer. This
indicates that neither the middle-layer material nor the encapsulation method has a
significant impact on the growth of E. coli. The detailed iteration process is shown in
the "Notebook" section.

This process reflects our cycle from one "Learn" phase to the next "Design" phase, and
from one cycle to the subsequent cycle, embodying our "Engineering Success".

Cycle 5: Evaluation of in Vitro Basic Gene Leakage of Engineered
Bacteria-Hydrogel

**## Design**

We designed a simple and reliable in vitro experiment to quantitatively evaluate the gene
leakage level of our hydrogel encapsulation system in a simulated intestinal basal
environment.

1.Environmental simulation : We chose to prepare simulated intestinal fluid ( SIF ), and
its key parameters included pH 6.8 and two key components : bile salt ( a surfactant
that can destroy membrane structure ) and trypsin ( a protease mixture ). This creates a
basic chemical stress environment.

2.Static conditions : The experiment was carried out under anaerobic, 37 ° C static
conditions to focus on evaluating the intrinsic stability of the material / strain and
excluding complex variables such as fluid dynamics.

3.Key separation steps : Use a 0.22 μm filter to filter the soaking solution to ensure
that we detect ' leaky ' genetic material ( free DNA, subcellular particles ), not
complete bacteria.

4.Sensitive detection : qPCR absolute quantification of specific genes ( such as
resistance genes ) on our engineering plasmids was used to provide highly sensitive and
quantifiable leakage data.

**## Build**

Materials preparation : Synthesis and purification of our engineered strains. Preparation
of hydrogel microspheres encapsulating engineering bacteria. At the same time, a blank
hydrogel without bacteria was prepared as a control.

Reagent preparation : The simulated intestinal fluid ( pH 6.8 ) containing 0.5 % bile
salt and 1.0 % trypsin was prepared and sterilized according to the Chinese
Pharmacopoeia. Preparation of qPCR reagents : specific primers / probes for the target
gene, purified plasmid DNA for the standard curve.

Establish a culture system : Hydrogel microspheres were incubated with simulated
intestinal fluid in a sterile centrifuge tube. The culture tube was placed in an
anaerobic tank filled with anaerobic mixture to simulate the intestinal hypoxia
environment.

**## Test**

Experiment execution and data acquisition :

1.Exposure and sampling : Parallel samples were taken from the anaerobic tank at the set
time points ( days 1,3, and 7 ).

2.Sample treatment : After violent vortex, the soaking solution was taken and passed
through a 0.22 μm filter. The collected filtrate represents the ' leakable component '.

3.DNA extraction and qPCR : Total DNA was extracted from the filtrate, and the qPCR
program was run to generate a standard curve using a plasmid standard with a known
concentration.

4.Data analysis : The Ct value of qPCR was substituted into the standard curve to
calculate the absolute copy number / mL of the target gene in each sample. The data of
the experimental group were statistically compared with the negative control group.

**## Learn**

The above experimental results will determine our next optimization and improvement of
the hydrogel.

If the level of gene leakage is extremely low : This indicates that our hydrogel system
has excellent closure under basic conditions. Based on this positive result, we will
advance to more complex tests ( such as horizontal gene transfer assessment ) to
challenge the safety of the system in more extreme environments.

If a significant leak is detected : We must analyze the leak pattern to determine the
root cause. In the case of early sudden leakage, it indicates that the physical
structure of the hydrogel is defective, and it is necessary to return to the Design
stage to optimize the material formulation. If a leak that continues to rise over time
is detected, it points to the cracking of the internal engineering bacteria. It is
necessary to return to the Build stage to transform the strain and enhance its
stability.

Regardless of the results, this experiment provides us with a clear direction for
improvement, ensuring that each iteration of the project is based on experimental
evidence.

References

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[2] Su Zekun.Design, preparation and evaluation of adhesive hydrogel coated
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10.26991 / d.cnki.gdllu.2024.000837.

[3] Zhao Q, Chen X, Ji W, Zhang J, Li X, Liu Z, Wang W, Liu H, Wang Y, Nan B, Li X, Wang
Y, Liu J. From lab to table: Recent advances in the application of sodium alginate-based
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10.1016/j.foodres.2025.116843. Epub 2025 Jun 9. PMID: 40597545.

[4] Chen Tiantian, Wang Congtian, Liu Wanxiang. et al. Preparation and drug release of
pH-responsive cellulose nanofibrils / sodium alginate based hydrogels [ J ]. Chinese
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[5] Xu Bin, Duan Cuimi, Hao Tong, et al. Preparation of chitosan hydrogels and their
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: 121-124. DOI : 10.19529 / j.cnki.1672-6278.2008.02.014.

[6] Shiraki M, Endo T, Saito T. Fermentative production of (R)-(−)-3-hydroxybutyrate
using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant
Escherichia coli. J Biosci Bioeng. 2006;102:529–34.

[7] De Roo G, Kellerhals MB, Ren Q, Witholt B, Kessler B (2002) Production of chiral
R-3-hydroxyalkanoic acids and R-3-hydroxyalkanoic acid methylesters via hydrolytic
degradation of polyhydroxyalkanoate synthesized by Pseudomonas. Biotechnol Bioeng
77:717–722

[8] Liu Q, Ouyang S-P, Chung A, Wu Q, Chen GQ. Microbial production of R-3-hydroxybutyric
acid by recombinant E. coli harboring genes of phbA, phbB, and tesB. Appl Microbiol
Biotechnol. 2007;76:811–8.

[9] Matsumoto, K.i., Okei, T., Honma..., I. 2013. Efficient (R)-3-hydroxybutyrate
production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase.
Applied Microbiology & Biotechnology, 97(1), 439-439.

[10] LIAO Junqi, HUANG Haining, CHEN Yinguang. CO2 promotes the conjunctive transfer of
multiresistance genes by facilitating cellular contact and plasmid transfer [J].
Environment International, 2019, 129:333-342.

[11] ZHANG Ye,GU A Z,CEN Tianyu,et al. Subinhibitory concentrations of heavy metals
facilitate the horizontal transfer of plasmid-mediated antibiotic resistance genes in
water environment[J]. Environmental Pollution,2018,237:74-82.

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# Experiments

Construction of Engineered Bacteria Transformation and BHB Synthesis Pathway1

I. Preparation of Escherichia coli Competent Cells and Transformation of Plasmid DNA
II. Extraction of Plasmid DNA
III. Detection of DNA by Agarose Gel Electrophoresis
IV. PCR Gene Amplification and Product Recovery
V. Linearized Plasmid Vectors
VI. DNA Recombination
VII. Colony PCR
VIII. Quantitative Detection of BHB

Preparation and Performance Testing of Hydrogels2

I. Preparation of Hydrogel Core and Bacterial Encapsulation
II. Preparation of the Middle Adhesive Layer of Hydrogel
III. Bacterial Viability and Biocompatibility Test (Determination of Growth Curve)
IV. Verification of Middle Layer Adhesion Performance
V. Preparation of the Hydrogel Outer Layer (pH-Responsive Layer)
VI. Verification of the pH-Responsive Performance of the Outer Layer
VII. Assessment of Baseline Gene Leakage from Engineered Bacteria-Hydrogel System

References3

Construction of Engineered Bacteria Transformation and BHB Synthesis Pathway

**## I. Preparation of Escherichia coli Competent Cells and Transformation of Plasmid DNA**

1. Experimental Objectives:

By preparing Escherichia coli competent cells and transferring the recombinant plasmid into them, the replication and amplification of plasmid DNA can be achieved, facilitating the extraction of plasmid DNA for subsequent experiments.

2. Experimental Principles:

The state in which bacteria are receptive to absorbing exogenous DNA is called competence. Transformation refers to the process of introducing plasmid DNA or its vector-constructed recombinant into bacteria. The principle is that bacteria are in a 0°C, low-salt hypotonic solution environment, and the cells expand and become spherical. The DNA in the transformation mixture forms a hydroxyl-calcium-phosphate DNA complex that adheres to the cell surface. After a short heat shock at 42°C, the cells absorb the DNA complex. The bacteria are placed in a non-selective medium and incubated for a period of time to allow the new phenotype acquired during the transformation process to be expressed. Then, the bacterial culture is spread on a selective medium.

3. Instruments, Materials and Reagents:

Instruments: Ultra-clean bench, low-temperature centrifuge, constant temperature shaker, water bath with water jacket, constant temperature water bath, autoclave;

Materials: Escherichia coli, plasmid DNA, CaCl2, peptone, yeast extract, NaCl, NaOH, agar, ampicillin, kanamycin, 50 mL centrifuge tube, test tube, petri dish, beaker, etc.;

Reagents: 0.1 mol/L CaCl2 solution, LB liquid medium, LB solid medium.

4. Experimental Protocols:

(1) Take 100 μL of competent Escherichia coli from the -80°C refrigerator and place it on ice to rapidly thaw. Add 10 μL of recombinant plasmid DNA within 8 minutes and gently tap the bottom of the centrifuge tube to mix (avoid up-and-down shaking). Incubate at 4°C for 25 minutes.

(2) Perform a 42-second heat shock at 42°C, then place the cells back on ice for 5 minutes.

(3) Add 700 μL of sterile LB medium without antibiotics to the tube, gently mix, and incubate at 37°C, 200 rpm shaker for 60 minutes to restore.

(4) Take 100 μL of the bacterial suspension and spread it on LB medium containing ampicillin and LB medium containing kanamycin (for low-concentration cultivation); centrifuge at 5000 rpm for 1 minute to collect the bacteria, retain about 100 μL of the supernatant, gently tap to resuspend the bacteria, and spread it on LB medium containing ampicillin and LB medium containing kanamycin (for high-concentration cultivation).

(5) Invert the plates and incubate in a 37°C incubator overnight, with a total incubation time of at least 15 hours at 37°C.

(6) Take 200 μL of competent cells and mix with 2 μL of plasmid DNA on ice for 30 minutes.

(7) Place the tube in a 42°C water bath for 90 seconds.

(8) Place on ice for 2 minutes.

(9) Add 800 μL of LB liquid medium to each tube, incubate at 37°C shaker at 150 r/min for 1 hour.

(10) Spread 100 μL of the recovered cells on LB petri dishes containing ampicillin and normal flat placement for 30 minutes. Similarly, spread 100 μL of the recovered cells on LB petri dishes containing kanamycin and normal flat placement for 30 minutes.

(11) Invert the plates and incubate at 37°C for 12-16 hours. Colony formation will occur.

**## II. Extraction of Plasmid DNA**

1. Experimental Objectives:

To extract the replicated and amplified plasmid DNA from the bacterial cells for the construction of recombinant plasmids.

2. Experimental Principles:

The alkaline lysis method for plasmid extraction is based on the topological differences between covalently closed circular plasmid DNA and linear chromosomal DNA. Within the narrow pH range of 12.0-12.5, the double helix structure of linear DNA unwinds and denatures. Although the hydrogen bonds of covalently closed circular plasmid DNA are broken under such conditions, the two complementary strands remain tightly intertwined. When pH 4.8 potassium acetate high-salt buffer is added to restore the pH to neutral, the covalently closed circular plasmid DNA rapidly and accurately renatures due to the two complementary strands remaining together, while the linear chromosomal DNA, with its two complementary strands completely separated, renatures less rapidly and accurately, entangling to form a network structure. Through centrifugation, the chromosomal DNA, along with unstable large-molecule RNA, protein-SDS complexes, etc., precipitates and is removed.

3. Instruments, Materials and Reagents:

Instruments: Constant temperature shaker, benchtop centrifuge, autoclave, vortex oscillator;

Materials: E. coli containing plasmids, Tris, EDTA, glucose, NaOH, SDS, KAc, HAc, HCl, Tris-saturated phenol, chloroform, isopropanol, ethanol, RNase A, kanamycin, ampicillin, sucrose

Reagents: Solution I (50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA), Solution II (0.4 M NaOH and 2% SDS mixed in equal volume before use), Solution III (5 M KAc 60 mL, 11.5 mL HAc,28.5 mL water), TE buffer (10 mM Tris-HCl, 1 mM EDTA), 0.5 M EDTA, chloroform-isopropanol, Tris-saturated phenol-chloroform-isopropanol, 70% ethanol, RNase A.

4. Experimental Protocols:

(1) Add 3 μL of antibiotic (final concentration 50 μg/mL) to 3 mL of LB liquid medium, then inoculate a single colony of E. coli containing plasmids and shake at 37°C overnight.

(2) Transfer the overnight culture to a 1.5 mL centrifuge tube, centrifuge at 4000 r/min for 1 min, and remove the culture medium thoroughly (otherwise, it may affect the enzymatic digestion reaction). Collect all the bacterial cells in a small tube.

(3) Add 100 μL of Solution I to the centrifuge tube containing the bacterial cells, vortex to suspend the bacterial pellet, and incubate at room temperature for 10 min.

(4) Add 200 μL of Solution II, gently mix the contents, and when the solution becomes clear, add Solution III (do not use a vortex oscillator; the lysis time should not exceed 5 min).

(5) Add 150 μL of Solution III (pre-cooled on ice), tightly close the tube, and gently mix several times. Place on ice for 15 min to allow the plasmid DNA to renature (do not use a vortex oscillator).

(6) Centrifuge at 12000 r/min for 10 min, and transfer the supernatant to another 1.5 mL centrifuge tube.

(7) Add an equal volume of phenol-chloroform-isopropanol to the supernatant to remove proteins and lipids, vortex to mix, and centrifuge at 12000 r/min for 5 min. Transfer the supernatant to another 1.5 mL centrifuge tube.

(8) Add an equal volume of chloroform-isopropanol to the supernatant to remove trace phenol and lipids, vortex to mix, and centrifuge at 12000 r/min for 5 min. Transfer the supernatant to another 1.5 mL centrifuge tube.

(9) Add two volumes of absolute ethanol to the supernatant, vortex to mix, and incubate at room temperature for 30 min (to precipitate the DNA). Centrifuge at 12000 r/min for 10 minutes and discard the supernatant.

(10) Wash the plasmid DNA precipitate twice with 1 mL of 70% ethanol (vigorously shake to mix well and centrifuge at 12000 r/min for 5 minutes each time), discard the supernatant and air dry.

(11) Add 20 μL of RTE (TE buffer containing 20 μg/mL pancreatic RNAse to degrade RNA) to completely dissolve the plasmid DNA and store at -20°C.

**## III. Detection of DNA by Agarose Gel Electrophoresis**

1. Experimental Objectives:

To detect the status of the target DNA fragment through agarose gel electrophoresis.

2. Experimental Principles:

When DNA molecules move in agarose gel, there are both electrostatic effects and molecular sieving effects. DNA molecules carry a negative charge in a pH solution higher than their isoelectric point and move towards the positive electrode in an electric field. Due to the repetitive nature of the sugar-phosphate backbone in structure, double-stranded DNA molecules of the same quantity carry almost the same amount of electrostatic charge and thus move towards the positive electrode at the same rate. Under a certain electric field intensity, the migration rate of DNA molecules depends on the molecular sieving effect, that is, the size and configuration of the DNA molecules themselves. DNA fragments with different relative molecular masses have different migration rates and can be separated. The migration rate of DNA molecules is inversely proportional to the logarithm of their relative molecular mass. Agarose gel electrophoresis can not only separate DNA with different relative molecular masses but also separate DNA molecules with the same relative molecular mass but different configurations, such as supercoiled covalently closed circular plasmid DNA, open circular plasmid DNA, and linear plasmid DNA. These three have different migration rates in agarose gel electrophoresis, so after electrophoresis, they may appear as three bands: supercoiled plasmid DNA migrates the fastest, followed by linear plasmid DNA, and then open circular plasmid DNA.

3. Instruments, Materials and Reagents:

Instruments: Agarose gel electrophoresis system, ultraviolet transilluminator;

Materials: Plasmid DNA or DNA fragments, agarose, boric acid, Tris, EDTA, bromophenol blue, sucrose, GoldView™;

Reagents: 1×TAE, 6×Loading buffer.

4. Experimental Protocols:

(1) Dissolve 1 g of agarose in 100 mL of 1×TAE buffer in a conical flask to prepare the agarose gel.

(2) Heat the mixture in a microwave until the agarose is completely dissolved. After boiling, gently shake the conical flask to ensure uniform dissolution. When the solution cools slightly, add 10 μL of GoldView™ nucleic acid dye.

(3) Pour the agarose solution into the gel preparation plate and let it solidify at room temperature.

(4) After solidification, carefully remove the comb, and place the gel (together with the preparation plate) into the electrophoresis tank. Add 1×TAE buffer to the electrophoresis tank until it covers the gel.

(5) Add 10 μL of 6×loading buffer to each sample. Load the samples into the gel, and add 5000 bp DNA standard as a size reference.

(6) Run the gel at 80 - 120 volts. After electrophoresis, observe the gel under ultraviolet light and take a picture of the result.

5. Reflection and Optimization:

According to the size of the DNA to be separated, we explored the appropriate percentage of agarose in the gel:

|  |  |
| --- | --- |
| Agarose gel concentration/% | Effective separation range of linear DNA/kb |
| 0.3 | 5-60 |
| 0.6 | 1-20 |
| 0.7 | 0.8-10 |
| 0.9 | 0.5-7 |
| 1.2 | 0.4-6 |
| 1.5 | 0.2-4 |
| 2.0 | 0.1-3 |

**## IV. PCR Gene Amplification and Product Recovery**

1. Experimental Objectives:

To amplify the target gene fragment using PCR and recover it to provide materials for the construction of recombinant plasmids.

2. Experimental Principles:

The principle of polymerase chain reaction (PCR) is similar to the natural replication process of DNA. By using a pair of oligonucleotide primers complementary to the two sides of the DNA fragment to be amplified, after several cycles of denaturation, annealing and extension, the DNA is amplified 2n times. A typical PCR reaction system includes: DNA template, reaction buffer, dNTP, MgCl2, a pair of synthetic DNA primers, and Taq enzyme. Denaturation: Heating the template DNA at high temperature to break the hydrogen bonds between the two strands, forming two single strands, which is the denaturation stage. Annealing: Lowering the solution temperature to 50-60°C, allowing the template DNA to bind to the primers according to the principle of base complementarity, which is the annealing stage. Extension: Raising the reaction temperature, Taq enzyme uses the single-stranded DNA as a template and, guided by the primers, replicates the complementary DNA in the 5'→3' direction using the four dNTPs in the reaction mixture, which is the extension stage. The above three steps constitute one cycle, namely the high-temperature denaturation, low-temperature annealing, and medium-temperature extension stages. Theoretically, within a certain number of cycles, the amount of DNA in the sample should double after each cycle, and the newly formed strands can serve as templates for the next round of cycles. After 25-30 cycles, the DNA can be amplified 106-109 times. Subsequently, the PCR products are recovered using a PCR product rapid gel recovery kit.

3. Instruments, Materials and Reagents:

Instruments: PCR thermal cycler, agarose gel electrophoresis system, ultraviolet transilluminator;

Materials: DNA template, dNTP mixture, primer pair, Taq enzyme, PCR tubes, pipette tips, centrifuge tubes, agarose, HAc, Tris, EDTA, bromophenol blue, sucrose, GoldView™, ethanol, PCR product rapid gel recovery kit;

Reagents: dNTP mixture, Taq enzyme, DNA template, primer pair, 1×TAE, 6×Loading buffer, PCR buffer.

Target gene and primer sequences:

|  |  |  |  |
| --- | --- | --- | --- |
| Target Gene | Sequence of Target Gene | Primer (F) | Primer (R) |
| pcT | ATGAGAAAAGTAGAAATCATTACAGCTGAACAAGCAGCTCAGCTCGTAAAAGACAACGACACGATTACGTCTATCGGCTTTGTCAGCAGCGCCCATCCGGAAGCACTGACCAAAGCTTTGGAAAAACGGTTCCTGGACACGAACACCCCGCAGAACTTGACCTACATCTATGCAGGCTCTCAGGGCAAACGCGATGGCCGTGCCGCTGAACATCTGGCACACACAGGCCTTTTGAAACGCGCCATCATCGGTCACTGGCAGACTGTACCGGCTATCGGTAAACTGGCTGTCGAAAACAAGATTGAAGCTTACAACTTCTCGCAGGGCACGTTGGTCCACTGGTTCCGCGCCTTGGCAGGTCATAAGCTCGGCGTCTTCACCGACATCGGTCTGGAAACTTTCCTCGATCCCCGTCAGCTCGGCGGCAAGCTCAATGACGTAACCAAAGAAGACCTCGTCAAACTGATCGAAGTCGATGGTCATGAACAGCTTTTCTACCCGACCTTCCCGGTCAACGTAGCTTTCCTCCGCGGTACGTATGCTGATGAATCCGGCAATATCACCATGGACGAAGAAATCGGGCCTTTCGAAAGCACTTCCGTAGCCCAGGCCGTTCACAACTGTGGCGGTAAAGTCGTCGTCCAGGTCAAAGACGTCGTCGCTCACGGCAGCCTCGACCCGCGCATGGTCAAGATCCCTGGCATCTATGTCGACTACGTCGTCGTAGCAGCTCCGGAAGACCATCAGCAGACGTATGACTGCGAATACGATCCGTCCCTCAGCGGTGAACATCGTGCTCCTGAAGGCGCTACCGATGCAGCTCTCCCCATGAGCGCTAAGAAAATCATCGGCCGCCGCGGCGCTTTGGAATTGACTGAAAACGCTGTCGTCAACCTCGGCGTCGGTGCTCCGGAATACGTTGCTTCTGTTGCCGGTGAAGAAGGTATCGCCGATACCATTACCCTGACCGTCGAAGGTGGCGCCATCGGTGGCGTACCGCAGGGCGGTGCCCGCTTCGGTTCGTCCCGCAATGCCGATGCCATCATCGACCACACCTATCAGTTCGACTTCTACGATGGCGGCGGTCTGGACATCGCTTACCTCGGCCTGGCCCAGTGCGATGGCTCGGGCAACATCAACGTCAGCAAGTTCGGTACTAACGTTGCCGGCTGCGGCGGTTTCCCCAACATTTCCCAGCAGACACCGAATGTTTACTTCTGCGGCACCTTCACGGCTGGCGGCTTGAAAATCGCTGTCGAAGACGGCAAAGTCAAGATCCTCCAGGAAGGCAAAGCCAAGAAGTTCATCAAAGCTGTCGACCAGATCACTTTCAACGGTTCCTATGCAGCCCGCAACGGCAAACACGTTCTCTACATCACAGAACGCTGCGTATTTGAACTGACCAAAGAAGGCTTGAAACTCATCGAAGTCGCACCGGGCATCGATATTGAAAAAGATATCCTCGCTCACATGGACTTCAAGCCGATCATTGATAATCCGAAACTCATGGATGCCCGCCTCTTCCAGGACGGTCCCATGGGACTGAAAAAATAA | TGGTGGTGGTGGTGCTCGAGATGAGAAAAGTAGAAATCATTACAGCTGAACAAG | CGTCGACAAGCTTGCGGCCGCTTATTTTTTCAGTCCCATGGGACCG |
| phaA | CTACACACGCTCAATAGCAAAAGCCGTACCGCCGCCAGTACCATGACACAAAGCCGCCAGCCCCAAACTCTTATCGTGTTGTTCCAAAGCATTAAGTAAAGTCACAACAATGCGGGAACCGGAAGCGCCAATAGGGTGTCCGAGAGCGATCGCCCCACCATTAACATTCAACTTATCTTGAGAGACCCCCAACATTGTATCAAATAACAAGGTACTAACTGCAAAAGCCTCGTTATTTTCAAACAAATCGAAATCATTAACCGACATTTTCAGGCGACCCAGTAACTTCTGAGTAGCCAAAATCGGCACTTCTGGAAACCGCCAACTCTCACCACCAGCCCAAGCGCCGCCCAGCACTTTAGCCAGAGGTTTCAAACCATACTTTTGTACAGCAGCCTCACTAGCCAAAATAATCGCCGCCGCACCATCAGAAATTTGGGAACTATTCCCAGCAGTTAAGACCCCATCTTTCTGAAAAGCCGGACGCAACTTAGCCAAACTTTCCAAAGTAGTCTCAGCGCGGATACCCTCATCAGTATCAATCACCTGAGTTCCCTTACGGCTAGACACCTCAATGGGGGAGATTTCATTTTTAAACAATCCTTTTTCCGTAGCTTCCGCCGCGCGTTTTTGGGAGTAAAAAGCCACCTCGTCCAAAGCCTGACGACTGAAACCATGTTCAGCCGCCAAACGTTCAGTTTCATCCCCCATACCGTCTCCGGTAGTAGAGTCTGTTAAACCATCATACAGCAGAATATCCATCAACTGTTCTGGGGCTCCCATAAGGAACTTATAACCCCATCTAGCGCGGTGAGACAAAAAGAACCCTGTTTGCGACATACACTCAGTCCCTCCTGCCAGCACTAGATCAGCCTCTCCAGCCTTAATAGTGTAGCAGCCATTAATCAGACTCATCATAGCAGAAGAACAAACCATATCAATGGCATATCCATCGACTGTGGCGGGTATACCTGCTTTCAAAGCCGCTTGACGGGGTATTAGCTGTCCGTGTCCTGCTCGCAACACATTTCCAAAAATATAAAGATCCAGATTTTCACCGGAAACCCCAGCTTTCGCCAATGCTGCTTTCATAGCATGAGCGCCTAGGTCAGCCGGAGAAAACCCAGATAACCCCCCCCCAAACCTACCTAGGGGGGTGCGTGTTGCGGAGACGATATAAACTTCTGTCAT | TGGTGGTGGTGGTGCTCGAGATGACAGAAGTTTATATCGTCTCCGC | CGTCGACAAGCTTGCGGCCGCCTACACACGCTCAATAGCAAAAGC |
| phaB | TCAGCCCATATGCAGGCCGCCGTTGAGCGAGAAGTCGGCGCCGGTCGAGAAACCGGACTCCTCCGACGACAACCAGGCGCAGATCGAGGCGATCTCTTCCGGCAGGCCCAGGCGCTTGACCGGGATCGTCGCGACGATCTTGTCGAGCACGTCCTGGCGGATCGCCTTGACCATGTCGGTGGCGATATAGCCCGGAGAGACCGTGTTGACGGTCACGCCCTTGGTCGCCACTTCCTGCGCCAGTGCCATGGTGAAGCCATGCAGGCCGGCCTTGGCGGTGGAGTAGTTGGTCTGGCCGAACTGGCCCTTCTGCCCGTTCACCGACGAGATGTTGACGATGCGGCCCCAGCCACGGTCGGCCATGCCGTCGATCACCTGCTTGGTGACGTTGAACAGCGAGGTCAGGTTGGTGTCGATCACCGCATCCCAGTCGGCGCGGGTCATCTTGCGGAACACCACGTCGCGGGTGATACCGGCGTTGTTGATCAGCACATCAACCTCGCCGACCTCGGACTTGACCTTGTCGAATGCGGTCTTGGTCGAGTCCCAGTCAGCCACATTGCCTTCCGAGGCAATGAAATCGAAGCCCAGGGCCTTCTGCTGCTCCAGCCACTTTTCGCGGCGCGGCGAGTTGGGGCCGCAACCGGCCACCACACGAAAGCCATCCTTGGCCAGCCGCTGGCAAATGGCGGTTCCGATACCACCCATGCCGCCGGTCACATACGCAATGCGCTGAGTCAT | TGGTGGTGGTGGTGCTCGAGATGACTCAGCGCATTGCG | CGTCGACAAGCTTGCGGCCGCTCAGCCCATATGCAGGCCGC |
| rplO | ATGCGTTTAAATACTCTGTCTCCGGCCGAAGGCTCCAAAAAGGCGGGTAAACGCCTGGGTCGTGGTATCGGTTCTGGCCTCGGTAAAACCGGTGGTCGTGGTCACAAAGGTCAGAAGTCTCGTTCTGGCGGTGGCGTACGTCGCGGTTTCGAGGGTGGTCAGATGCCTCTGTACCGTCGTCTGCCGAAATTCGGCTTCACTTCTCGTAAAGCAGCGATTACAGCCGAAATTCGTCTGTCTGACCTGGCTAAAGTAGAAGGCGGTGTAGTAGACCTGAACACGCTGAAAGCGGCTAACATTATCGGTATCCAGATCGAGTTCGCGAAAGTGATCCTGGCTGGCGAAGTAACGACTCCGGTAACTGTTCGTGGCCTGCGTGTTACTAAAGGCGCTCGTGCTGCTATCGAAGCTGCTGGCGGTAAAATCGAGGAATAA | CATCACCACAGCCAGGATCCATGCGTTTAAATACTCTGTCTCCGG | CAGGCGCGCCGAGCTCTTATTCCTCGATTTTACCGCCAGCA |
| SUMO-Tag | TCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACCCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTGGT | CTTGTCGACGGAGCTCTCGGACTCAGAAGTCAATCAAGAAGC | AGCAAATGGGTCGCGGATCCACCAATCTGTTCTCTGTGAGCC |

4.Experimental Protocols:

(1) Take a PCR tube (0.1 mL) and place it on ice. Preheat the PCR machine in advance.

(2) Mix the following reagents in the PCR tube on ice:

2×UKOD Master Mix: 25 μL

Forward primer: 1 μL

Reverse primer: 1 μL

Template DNA: 15 ng

Make up to 50 μl with double-distilled water.

(3) Briefly centrifuge the PCR tube to mix the reagents and remove any bubbles.

(4) Place the PCR tube in the PCR machine and set the following program:

98℃ for 3 minutes

98℃ for 10 seconds

68℃ for 15 seconds

68℃ for 5 seconds (30 cycles)

68℃ for 5 minutes

4℃ for an indefinite period

(5) Preheat the metal bath to 56℃.

(6) Under ultraviolet light, carefully cut out the gel slice containing the target DNA band with a clean surgical knife.

(7) Accurately weigh the gel slice and place it in a 2 mL microcentrifuge tube. Add 3 times the volume of solubilization buffer DD (if the gel weight is 100 mg, add 300 μL of solubilization buffer). If the gel concentration is greater than 2%, add 6 times the volume of DD.

(8) Incubate in a 56°C water bath for 10 minutes. Vortex every 2-3 minutes to accelerate dissolution.

(9) Add the solution obtained in the previous step to the adsorption column EC (place the adsorption column in a collection tube), and let it stand at room temperature for 1 minute. Centrifuge at 12,000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube.

(10) Add 500 μL of washing buffer WB (already containing absolute ethanol), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid. Repeat the operation.

(11) Place the adsorption column EC back into an empty collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much washing buffer as possible to avoid residual ethanol from the washing buffer inhibiting downstream reactions.

(12) Take out the adsorption column EC and place it in a clean centrifuge tube. Let it stand at room temperature for several minutes.

(13) Add elution buffer EB to the middle of the adsorption membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute. If a larger amount of DNA is needed, the obtained solution can be re-added to the adsorption column and centrifuged for 1 minute.

**## V. Linearized Plasmid Vectors**

1. Experimental Objectives:

To linearize the circular vector plasmid DNA by double digestion with two different restriction endonucleases at specific sites, generating two distinct ends, to achieve directional cloning and ensure that the exogenous fragment is inserted into the vector in the correct orientation, and to prepare the backbone vector for subsequent recombination reactions.

2. Experimental Principles:

Two restriction endonucleases recognize specific sequences on the plasmid vector and cleave the DNA backbone. When both recognition sites are cut, the circular plasmid is converted into a linear molecule with different sticky or blunt ends at each end. This asymmetric end structure effectively prevents self-ligation of the vector and forces the exogenous insert fragment to be inserted into the vector in the preset direction when it has a matching end, thereby achieving directional cloning and significantly improving the accuracy rate of positive clones. Selecting two enzymes that can work efficiently in the same buffer system (compatible buffer) is one of the keys to the success of this experiment.

3. Instruments, Materials and Reagents:

Instruments: Water bath or metal bath, microcentrifuge, vortex mixer, agarose gel electrophoresis system;

Materials: Circular plasmid DNA, XhoI Restriction Enzyme, NotI Restriction Enzyme, BamHI Restriction Enzyme, SacI Restriction Enzyme, restriction digestion buffer, sterile PCR tubes, 1.5 mL centrifuge tubes, pipette tips, agarose gel DNA recovery kit;

Reagents: ddH2O, 1×TAE, 6×DNA Loading buffer, DNA marker.

4.Experimental protocols:

Restriction Enzyme Digestion of Gel-Purified Vector (pRSFDuet-1)

(1) Take a PCR tube (0.1 mL) and place it on ice. Set up the restriction digestion reaction as follows:

DNA: 1μg

10× Restriction Enzyme Buffer: 5μL

BamHI Restriction Enzyme: 1μL

SacI Restriction Enzyme: 1μL

ddH₂O: add to a final volume of 50μL

(2) Briefly centrifuge the mixture to remove any air bubbles.

(3) Incubate at 37℃ for 5 - 15 minutes.

(4) Incubate at 65℃ for 20 minutes for heat inactivation.

Restriction Enzyme Digestion of Gel-Purified Vector (pET-21a)

(1)Take a PCR tube (0.1 mL) and place it on ice. Set up the restriction digestion reaction as follows:

DNA: 1μg

10× Restriction Enzyme Buffer: 5μL

XhoI Restriction Enzyme: 1μL

NotI Restriction Enzyme: 1μL

ddH₂O: add to a final volume of 50μL

(2) Briefly centrifuge the mixture to remove any air bubbles.

(3) Incubate at 37℃ for 5 - 15 minutes.

(4) Incubate at 65℃ for 20 minutes for heat inactivation.

**## VI. DNA Recombination**

1. Experimental Objectives:

To efficiently and precisely recombine the target DNA fragment into the linearized cloning vector through seamless cloning technology, construct the recombinant plasmid, and use it for subsequent transformation, amplification, and functional studies.

2. Experimental Principles:

Seamless cloning (also known as recombination cloning) is a cloning technique that does not rely on restriction endonucleases and ligases. Its principle is to utilize the exonuclease activity of DNA polymerase (such as Exnase II) to generate 15-25 bp complementary single-stranded ends at the termini of the cloning vector and the insert fragment. When the vector and the insert fragment are mixed in a certain ratio, these complementary single-stranded ends anneal to form a circular DNA molecule with a gap. Subsequently, this mixture is directly transformed into competent cells, and the repair mechanism within the cells automatically fills the gap to form a complete and biologically active recombinant plasmid. This method overcomes the limitations of the traditional enzyme digestion-ligation method, allowing any fragment to be inserted into any site of the vector as long as homologous arms are designed.

3.Experimental Design:

Use plasmids pRSFDuet-1 and pET-Duet-1 to ligate the rplO, phaA and phaB, and pcT gene fragments respectively, and construct recombinant plasmids. The plasmid and recombinant plasmid maps are as follows:

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5911/experiment/prsfduet-1.webp) | ![](https://static.igem.wiki/teams/5911/experiment/prsfduet-1-rplo-a-2.webp) |
| ![](https://static.igem.wiki/teams/5911/experiment/pet-duet-1.webp) | ![](https://static.igem.wiki/teams/5911/experiment/pet-duet-1-t-b-2.webp) |
| Figure 1.the map of two plasmid backbones and recombinant plasmids (a.pRSFDuet-1; b.pRSFDuet-1-rplO&phaA c.pET-Duet-1; d.pET-Duet-1-pcT&phaB) | |

4. Instruments, Materials and Reagents:

Instruments: Constant temperature shaker, constant temperature water bath, water bath incubator, benchtop centrifuge, low-temperature centrifuge, laminar flow hood, ultraviolet transilluminator;

Materials: Biomed™ Seamless Cloning Kit CL116, linearized cloning vector, target gene DNA fragment, sterile PCR tubes, pipette tips, 1.5 mL centrifuge tubes, Escherichia coli DH5α competent cells;

Reagents: Components in the kit, sterile ultrapure water.

5. Experimental Protocols:

(1) Prepare the reaction system according to the table below (0.2 mL PCR tube):

|  |  |
| --- | --- |
| Component | Volume |
| PCR product (50-100 ng/μL) | XμL |
| Linearized vector (50-100 ng/μL) | YμL |
| 2×Seamless Cloning Mix | 5μL |
| Make up to total volume with water | 10μL |

(2) Gently mix and centrifuge for a few seconds. Incubate at 50℃ for 15 minutes on a PCR instrument. After the reaction, place the tube on ice for transformation experiments.

(3) Take the ligation product and add it to freshly thawed competent cells, gently mix, and incubate on ice for 20-30 minutes.

(4) Heat shock at 42℃ for 90 seconds in a water bath without disturbing the water surface. Immediately place the tube in an ice bath for 2 minutes after heat shock.

(5) Add 500 μL of LB medium to the tube and incubate in a 37℃ shaking incubator at approximately 200 rpm for 60 minutes.

(6) Centrifuge at 4000 rpm for 1 minute, discard the supernatant, and retain 100-200 μL. Resuspend the bacterial pellet gently by pipetting. Take half of the bacterial suspension and spread it on an LB solid medium plate containing antibiotics. After the liquid is absorbed, invert the plate and incubate at 37℃ overnight.

6. Reflection and Optimization:

(1) The amount of vector is generally better at 50-100 ng. The molar ratio of vector to fragment is 1:1 to 1:3. When the fragment is less than 200 bp, the amount of fragment can be increased to five times that of the vector.

(2) The reaction time at 50℃ should not exceed 60 minutes.

**## VII. Colony PCR**

1. Experimental Objectives:

To verify the presence of plasmids in bacterial colonies through colony PCR without the need for DNA purification.

2. Experimental Principles:

Colony PCR is based on the property of high-temperature lysis of bacterial cells to release DNA. By designing universal primers for the vector and specific primers for the insert, the target DNA sequence can be directly amplified. The universal primers for the vector bind to the conserved regions on both sides of the multiple cloning site of the plasmid, while the specific primers for the insert bind within the insert fragment. The combination of these two types of primers can verify the orientation and integrity of the insert. The high-temperature treatment step in the reaction system rapidly lyses the bacterial cells, and the released DNA is amplified under the action of Taq enzyme. When the target DNA is present, the distance between the primers on both sides of the vector will produce an expected size of amplification product due to the length of the insert fragment.

3. Experimental Design:

In addition to the treatment group, empty vector control and untransformed colony control are set up.

4. Instruments, Materials and Reagents:

Instruments: PCR instrument, agarose gel electrophoresis system, ultraviolet transilluminator, microcentrifuge, super-clean bench;

Materials: Colonies to be screened, sterile PCR tubes, sterile toothpicks, pipette tips with filters, Taq enzyme and the corresponding 2×PCR Master Mix, specific primers, sterile ultrapure water;

Reagents: 2×PCR Master Mix, Forward Primer, Reverse Primer, sterile ultrapure water, 1×TAE electrophoresis buffer, DNA marker.

5. Experimental Protocols:

(1) Based on the number of colonies picked, prepare corresponding 1.5 mL microcentrifuge tubes and add 500 μL of LB broth containing kanamycin (1:1000). Use a pipette tip to pick a single colony and place it into the microcentrifuge tube. Incubate at 37°C.

(2) PCR Setup: Replace 1 μL of water with the bacterial culture. PCR Mixture (for Colony/Bacterial PCR):

|  |  |
| --- | --- |
| Components | Volume |
| ddH₂O | 4μL |
| Forward Primer | 0.5μL |
| Reverse Primer | 0.5μL |
| Taq Enzyme | 5μL |

(3) Mix the solution system thoroughly. If droplets adhere to the tube wall, perform a quick centrifugation.

Place into the PCR Instrument: (35 cycles)

95°C for 3 minutes

95°C for 30 seconds

55°C for 30 seconds

72°C for 90 seconds

72°C for 10 minutes

(4) After PCR is completed, perform gel electrophoresis to detect the products, and extract plasmids from the correct colonies.

**## VIII. Quantitative Detection of BHB**

1. Experimental Objectives:

To quantitatively detect the BHB concentration in the fermentation broth of engineered bacteria by enzyme kinetics methods to evaluate the metabolic engineering modification effect of the engineered bacteria.

2. Experimental Principles:

This experiment employs the specific reaction catalyzed by β-hydroxybutyric acid dehydrogenase. In a buffer environment with pH 8.5, BHB is oxidized by NAD+ to acetoacetic acid and NADH under the catalysis of β-hydroxybutyric acid dehydrogenase. The reaction equation is as follows: BHB+NAD+→acetoacetic acid+NADH+H+. The generated NADH has a characteristic absorption peak at 340nm. By dynamically monitoring the increase rate of absorbance (OD value) at 340nm with a microplate reader, this increase rate is directly proportional to the BHB concentration in the sample. By comparing the reaction rate of the standard solution, the accurate concentration of BHB in the sample can be calculated.

3. Instruments, Materials and Reagents:

Instruments: Microplate reader (with 340nm filter and constant temperature incubation function), micropipettes and associated tips, vortex mixer, timer, centrifuge, pH meter;

Materials: 96-well plates, 1.5mL/2mL centrifuge tubes;

Reagents: BHB detection buffer (containing Tris-HCl, pH ~8.5), NAD+ solution (high purity, ~30mM), β-hydroxybutyric acid dehydrogenase solution, BHB standard solution (concentration gradient, for example: 0, 0.5, 1.0, 2.0, 4.0, 8.0 mM), ddH2O.

4. Experimental Protocols:

(1) Sample pretreatment: Take the fermentation broth of the engineered bacteria, centrifuge at 12,000 rpm for 5 minutes, and take the supernatant. If the sample concentration is too high, it needs to be appropriately diluted with deionized water or PBS.

(2) Preparation of working solution: According to the number of samples to be tested and standards, mix BHB detection buffer, NAD+ solution and β-hydroxybutyric dehydrogenase solution in proportion to prepare the BHB detection working solution. It should be freshly prepared and used immediately, and kept on ice.

(3) Sample addition: Add samples in a 96-well plate according to the following scheme:

|  |  |
| --- | --- |
| Composition within the hole | Volume (μL) |
| BHB detection working solution | 90 |
| BHB standard or sample | 10 |
| Total volume | 100 |

Gently mix the solution.

(4) Kinetic Detection: Place the reaction plate in the microplate reader preheated to 37°C. Immediately start the kinetic monitoring program: Detection wavelength: 340nm (main wavelength), reference wavelength can be 410nm or not used. Detection mode: Kinetic Mode. Interval time: 10-30 seconds. Total monitoring time: 5-10 minutes. The microplate reader will automatically record the absorbance change curve at 340nm for each well over time.

(5) Data Calculation and Analysis: On the kinetic curve, select the linear growth phase (usually 1-5 minutes after the reaction starts). Calculate the change in absorbance per minute (ΔOD/min) for each well during this time period, which is the reaction rate. Plot a standard curve with the concentration of BHB standard as the x-axis (X) and the corresponding average ΔOD/min as the y-axis (Y). This curve is usually a straight line passing through the origin or can be fitted with a linear regression equation. Substitute the ΔOD/min value of the sample into the regression equation of the standard curve to calculate the concentration of BHB in the sample. If the sample has been diluted, multiply the calculated concentration by the corresponding dilution factor to obtain the actual BHB concentration in the original sample.

Preparation and Performance Testing of Hydrogels

**## I. Preparation of Hydrogel Core and Bacterial Encapsulation**

1. Experimental Objectives:

Prepare alginate core hydrogel beads encapsulated with Escherichia coli (E. coli) Nissle 1917, which will serve as the foundation for the subsequent multi-layer hydrogel structure and act as the inner nutrient layer.

2. Experimental Principles:

Sodium Alginate is a natural polysaccharide. When the G-units on its molecular chain come into contact with divalent cations (e.g., Ca²⁺), ionic cross-linking occurs, forming a "egg-box" gel with a three-dimensional network structure. This process is carried out under mild conditions, making it suitable for encapsulating viable bacteria without causing their death.

3. Instruments, Materials and Reagents:

Instruments: Ultra-clean workbench, Autoclave, Constant temperature shaker, Analytical balance, Pipette, Syringe, Petri dish, Erlenmeyer flask

Materials: Escherichia coli Nissle 1917 strain, Parafilm

Reagents: Tryptone, Yeast extract, Sodium chloride (NaCl), Sodium Alginate (SA), Calcium chloride (CaCl₂)

4. Experimental Protocols:

(1) Prepare 200 mL of LB liquid medium (without antibiotics) and sterilize it.

(2) Formulation: Dissolve 2 g of tryptone, 1 g of yeast extract, and 2 g of sodium chloride in deionized water, then bring the total volume to 200 mL.

(3) Weigh 2.5 g of sodium alginate and dissolve it in 47.5 mL of deionized water to prepare a 5 w.t.% sodium alginate solution. Sterilize it by autoclaving at 120°C for 20 minutes to ensure sterility.

(4) Weigh 5 g of calcium chloride and dissolve it in 100 mL of deionized water to prepare a 5 w.t.% CaCl₂ solution. Sterilize it by autoclaving at 120°C for 20 minutes to ensure sterility.

(5) Inoculate E. coli Nissle 1917 into the prepared medium, and incubate it in a constant temperature incubator at 37°C for 4–6 hours.

(6) Measure the absorbance value at 600 nm using a microplate reader. According to the standard that 1 OD contains 2×10⁸ CFU, take 1 OD of bacteria (2×10⁸ CFU).

(7) Inside the ultra-clean workbench, mix 20 mL of fresh bacterial culture with 20 mL of sodium alginate solution at a volume ratio of 1:1, resulting in a final sodium alginate concentration of 2.5 w.t.%.

(8) Inside the ultra-clean workbench, load the bacteria-sodium alginate pre-mixture into a syringe; transfer the CaCl₂ solution into a petri dish, ensuring the solution covers two-thirds of the dish.

(9) Drop the mixture from the syringe into the CaCl₂ solution and allow it to solidify for 15 minutes, forming bead-shaped droplets with a controllable diameter between 2.5 mm and 3 mm.

(10) Remove the solidified hydrogel cores from the CaCl₂ solution, place them in a new petri dish inside the ultra-clean workbench, seal the dish with parafilm, and store it in a refrigerator at 4℃.

5. Precautions and Reflections:

(1) All operations must be performed under sterile conditions to prevent contamination by miscellaneous bacteria.

(2) When dropping the mixture with a syringe, control the angle of the syringe needle to obtain hydrogel beads of uniform size and regular shape.

(3) The solidification time should not be too long; otherwise, the gel will become excessively hard, which affects subsequent coating, the diffusion of nutrients, and the overall preparation effect.

**## II. Preparation of the Middle Adhesive Layer of Hydrogel**

1. Experimental Objectives:

Wrap the core hydrogel beads with a composite coating of polydopamine-sodium alginate-polyacrylamide, which has strong adhesiveness and can prevent the passage of large DNA molecules.

2. Experimental Principles:

(1) Polydopamine (PDA): Dopamine can undergo self-polymerization under weakly alkaline conditions to form polydopamine with strong adhesiveness. It can bind to the functional groups of sodium alginate through Michael addition or Schiff base reaction.

(2) Polyacrylamide (PAM): Acrylamide (AM) undergoes free radical polymerization under the action of cross-linking agents, initiators, and catalysts to form a stable network.

(3) Secondary Cross-Linking: Using a cross-linking agent can activate the carboxyl groups of sodium alginate, forming more stable covalent bonds with the hydrazide groups on the polyacrylamide network, thereby enhancing the mechanical strength and compactness of the middle layer.

3. Instruments, Materials and Reagents:

Instruments: Ice packs, Magnetic stirrer, Analytical balance, pH meter,

Materials: Prepared hydrogel cores, Petri dishes, Erlenmeyer flasks, Graduated cylinders, Pipettes

Reagents: Sodium Alginate (SA), Dopamine Hydrochloride (DA), Sodium Hydroxide (NaOH), Acrylamide (AM), MES Buffer, NaCl, Cross-linking agent, Initiator, Catalyst

4. Experimental Protocols:

(1) Weigh 1 g of sodium alginate and dissolve it in 49 mL of deionized water to prepare a 2 w.t.% sodium alginate solution.

(2) Measure the pH of the sodium alginate solution using a pH meter, adjust the solution to weakly alkaline with 2 mol/L NaOH solution, and sterilize it by autoclaving at 120°C for 20 minutes to ensure sterility.

(3) Add 0.6 g of dopamine hydrochloride (DA) to the sodium alginate solution and stir at room temperature for 24 hours to obtain the PDA-SA solution.

(4) Under an ice-water bath (approximately 4°C), sequentially add 8 g of acrylamide (AM) and 30 mg of cross-linking agent to the above solution, and stir thoroughly until uniform.

(5) Add 60 mg of initiator and 60 μL of catalyst to the mixed solution, and mix thoroughly until uniform.

(6)Pour the above mixed solution into a clean petri dish, and immerse the hydrogel cores in it to form a thin shell layer.

(7) Accurately measure 50 mL of 0.2 M MES buffer and pour it into a clean beaker. Weigh 2.922 g of solid NaCl, add it to the above beaker, then add 30 mL of pure water, stir until the NaCl is completely dissolved, and bring the total volume to 100 mL.

(8) Weigh the cross-linking agent and catalyst, sequentially add them to the MES buffer, and dissolve and mix thoroughly.

(9) Immerse the coated hydrogel cores in the prepared MES buffer containing the cross-linking agent and catalyst to form covalent bonds and reinforce the middle layer. Maintain this state for 3 hours.

(10) Remove the reinforced hydrogel beads from the buffer, place them in a clean petri dish, seal the dish with parafilm, and store it in a refrigerator at 4℃.

5. Precautions:

(1) Acrylamide monomer is neurotoxic; gloves must be worn during operation, and weighing should be conducted in a fume hood.

(2) The cross-linking agent is prone to decomposition, so it should be prepared immediately before use to ensure the cross-linking effect.

(3) The polymerization reaction starts rapidly after adding the catalyst, so subsequent operations should be completed as soon as possible.

(4) The hydrogel cores should not be soaked in the hydrogel precursor solution for too long; otherwise, excessive cross-linking will increase brittleness and impair mechanical properties.

**## III. Bacterial Viability and Biocompatibility Test (Determination of Growth Curve)**

1. Experimental Objectives:

To verify that the components of each layer of the hydrogel and the preparation process have no negative impact on the activity of encapsulated Escherichia coli, and to demonstrate the biocompatibility of the materials.

2. Experimental Principles:

The hydrogel is broken by mechanical homogenization to release internal bacteria, and then a microplate reader is used to continuously monitor the absorbance of the culture at a wavelength of 600nm (OD₆₀₀). OD₆₀₀ is proportional to the bacterial concentration. By plotting the growth curve, the growth of bacteria in different treatment groups can be compared.

3. Instruments, Materials and Reagents:

Instruments: Microplate reader, tissue grinder, spectrophotometer, 96-well plate, centrifuge tube.

Materials: Hydrogel samples of each group (core, with middle layer), sterile LB medium.

Reagents: Stainless steel grinding beads, sodium alginate and other materials used for preparing hydrogels.

4. Experimental Proctocols:

(1) Put hydrogel samples of different groups into centrifuge tubes containing LB medium, add grinding beads for homogenization.

(2) Measure the OD₆₀₀ of the homogenate and adjust to the same initial concentration.

(3) Add 200μL samples into 96-well plates according to groups (A-L), with 5 replicate wells in each group.

The systems of each group are as follows:

Group A: LB medium

Group B: LB medium + 5% SA (1:1 mixture)

Group C: LB medium + 5% SA + CaCl₂ (curing agent) (system: 5mL LB + 5mL SA + 0.1mL CaCl₂)

Group D: LB medium + 5% SA + PDA solution (system: 5mL LB + 4.5mL SA + 0.5mL PDA)

Group E: LB medium + 5% SA + PDA solution + initiator (system: 5mL LB + 4.5mL SA + 0.5mL PDA + 10μL initiator)

Group F: LB medium + EcN

Group G: LB medium + 5% SA + EcN

Group H: LB medium + 5% SA + CaCl₂ (curing agent) + 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 after coating with middle layer

(4) Place the 96-well plate in a microplate reader, culture with continuous shaking at 37°C for 24 hours, and automatically measure OD₆₀₀ at intervals.

(5) For data processing, eliminate the maximum and minimum values, take the average, and draw the growth curve.

5. Precautions:

(1) The homogenization conditions must be consistent, and the grinding must be sufficient to ensure complete release of bacteria.

(2) The edge wells of the 96-well plate have a strong evaporation effect, so do not place our samples in them; fill them with PBS or sterile water instead.

(3) Sufficient control groups must be set up in the experiment (such as medium without bacteria, bacterial solution without materials, etc.) to accurately evaluate the impact.

**## IV. Verification of Middle Layer Adhesion Performance**

1. Experimental Objectives:

Quantitatively evaluate the adhesion ability of the hydrogel's middle layer coating to colorectal cancer cells (Caco-2 cells) and simulate the effect of the hydrogel adhering to and colonizing the intestinal tract.

2. Experimental Principles:

A monolayer of in vitro cultured Caco-2 cells is used to simulate the intestinal epithelium. Hydrogel beads are placed on the cell surface; under the shear force provided by a shaker, the adhesion strength is quantified by recording the number of gel beads that remain adhered at each time point under different shaking speeds. The stronger the adhesion, the less likely the beads are to move under shear force.

3. Instruments, Materials and Reagents:

Instruments：CO₂ incubator, Inverted microscope, Horizontal shaker, Digital camera

Materials: Culture flasks with a monolayer of Caco-2 cells, Empty culture flasks, Hydrogel cores (control group), Hydrogel beads coated with the middle layer

Reagents: Cell culture medium (high-glucose DMEM), PBS (Phosphate-Buffered Saline), Trypsin, etc.

4. Experimental Protocols:

(1) Culture and passage Caco-2 cells until a dense monolayer is formed at the bottom of the culture flask.

(2) Aspirate and discard the culture medium, then add 100 hydrogel cores and 100 hydrogel beads coated with the middle layer into two culture flasks with cell monolayers and one empty culture flask (control group), respectively.

(3) Invert the culture flasks on the shaker and take photos to record the initial position and state of all beads.

(4) Start the shaker (e.g., 50 rpm), take photos for recording every 5 minutes, and continue for 40 minutes.

(5) Change to different shaking speeds (e.g., 100 rpm, 150 rpm) and repeat the experiment.

(6) Analyze the photos: count the number of hydrogels still adhering to the cell layer at each time point (0, 5, 10, ..., 40 min) and calculate the adhesion ratio.

(7) Repeat the above experiment, calculate the average value, and evaluate the hydrogel's adhesion performance.

5. Precautions:

(1) When passaging Caco-2 cells, pipette and mix them evenly to avoid cell clumping, which may affect the flatness required for the experiment.

(2) Before the experiment, ensure that the cells are in good condition and the cell monolayer is intact.

**## V. Preparation of the Hydrogel Outer Layer (pH-Responsive Layer)**

1. Experimental Objectives:

Construct a calcium alginate film on the outermost layer. This layer can rapidly swell and rupture in the weakly alkaline environment of the intestinal tract, thereby exposing the adhesive middle layer.

2. Experimental Principles:

The "freeze-crosslinking" method is adopted. First, the gel beads coated with the middle layer are frozen to turn the internal water into ice crystals, which destroys part of the gel structure and increases pores. Then, the beads are immersed in a CaCl₂ solution for secondary crosslinking to form a denser calcium alginate outer layer. This layer is stable in the acidic gastric juice; in the weakly alkaline intestinal juice (pH 7.4), the carboxyl groups of the alginate become ionized, generating electrostatic repulsion. This causes the gel network to absorb water, swell, and eventually rupture.

3. Instruments, Materials and Reagents:

Instruments: -20°C refrigerator, magnetic stirrer.

Materials: Hydrogel beads coated with the middle layer.

Reagents: Calcium chloride (CaCl₂), sodium alginate (SA).

4. Experimental Protocols:

(1) Place the hydrogel beads with the middle layer in a -20°C environment for freezing for 5 hours.

(2) Quickly immerse the frozen samples in a 3 w.t.% CaCl₂ solution and stir at a low speed for 20 minutes.

(3) Take out the samples and rinse them with deionized water.

(4) Immerse the samples in a 1 w.t.% SA solution again and soak for 15 minutes to form a complete calcium alginate film on the surface.

(5) Take out the finished hydrogel, rinse it with PBS buffer, and store it in a humid environment for later use.

5. Notes:

(1) Ensure sufficient freezing time to completely freeze the interior of the beads.

(2) Maintain gentle stirring during the crosslinking and film-forming processes to ensure uniform reaction.

(3) The final product should be stored in a humid environment for at least 3 hours to prevent drying and water loss, and to facilitate better crosslinking of the outer layer.

**## VI. Verification of the pH-Responsive Performance of the Outer Layer**

1. Experimental Objectives:

Verify the swelling behavior of the hydrogel outer layer in environments with different pH values (simulated gastric juice and intestinal juice), and confirm that it possesses intestinal pH-responsive properties.

2. Experimental Principles:

Hydrogels absorb water and swell in solutions. Their swelling ratio is related to the crosslinking density of the gel network, hydrophilicity-hydrophobicity, and the environmental pH. By placing the hydrogel in PBS buffers with different pH values, periodically weighing its weight change, and calculating the swelling ratio, the pH-responsive properties of the hydrogel can be characterized. The formula is: Swelling Ratio = (Wₛ - W₀) / W₀ × 100%, where Wₛ is the weight after swelling and W₀ is the initial dry weight.

3. Instruments, Materials and Reagents:

Instruments: Analytical balance, constant temperature incubator, beakers, filter paper.

Materials: Complete three-layer structured hydrogel beads.

Reagents: PBS buffers (with pH values of 1.29, 3.64, 5.70, 7.06, 9.77, and 11.10 respectively).

4. Experimental Protocols:

(1) Dry the prepared hydrogel at room temperature for 3 hours, then weigh it to obtain the initial weight W₀.

(2) Immerse the samples in PBS buffers with pH values of 1.29, 3.64, 5.70, 7.06, 9.77, and 11.10 respectively.

(3) Place the samples in a 37°C constant temperature incubator. Take out the samples every 10 minutes, quickly blot the surface moisture with filter paper, then weigh them and record the weight as Wₛ.

(4) Continue the measurement until the weight remains basically unchanged (approximately 80 minutes).

(5) Calculate the swelling ratio at each time point according to the formula, and plot a swelling ratio-time curve.

5. Notes:

(1) When blotting the surface moisture, the operation should be quick and consistent to avoid human error.

(2) After each weighing, the solution should be replaced or its volume should be ensured to be sufficiently large to prevent the pH from changing due to leachables.

(3) The experimental environment (temperature, humidity) must be strictly controlled to ensure the repeatability of the results.

**## VII. Assessment of Baseline Gene Leakage from Engineered Bacteria-Hydrogel System**

1. Experimental Objectives:

To quantitatively evaluate whether the hydrogel encapsulation system exhibits baseline leakage of plasmid DNA from the engineered bacteria under static, simulated intestinal conditions, in the absence of external stressors.

2. Experimental Principles:

The hydrogel beads encapsulating engineered bacteria are incubated in Simulated Intestinal Fluid (SIF) under anaerobic conditions at 37°C. Any genetic material leaking from the beads into the surrounding fluid is collected by filtration (0.22 µm) to remove intact cells. The filtrate is then analyzed using absolute quantitative PCR (qPCR) targeting a specific gene on the plasmid, allowing for the detection and quantification of even low levels of free-floating DNA, indicating leakage.

3. Instruments, Materials and Reagents

Instruments: Anaerobic jar with gas exchange system、37°C Constant Temperature Incubator、Microcentrifuge、Vortex Mixer、qPCR Instrument、Biosafety Cabinet、0.22 µm Syringe Filters (sterile)

Materials:

Experimental Group: Hydrogel beads encapsulated with engineered bacteria.

Negative Control Group: Hydrogel beads without bacteria (blank).

Sterile 1.5 mL & 2 mL Microcentrifuge Tubes

Sterile Pipette Tips (with filter)

Reagents:

· Simulated Intestinal Fluid (SIF), pH 6.8:

 · 6.8 g/L KH₂PO₄

 · Adjusted with NaOH

 · 0.5% (w/v) Porcine Bile Salts

 · 1.0% (w/v) Pancreatin

 · (Sterilized by autoclaving, enzymes and bile salts added aseptically before use)

· DNA Extraction Kit (suitable for environmental samples/body fluids)

· qPCR Master Mix

· Forward and Reverse Primers (specific to the target gene on the plasmid, e.g., antibiotic resistance gene)

· Plasmid DNA Standard (for absolute quantification)

· Nuclease-Free Water

4. Experimental Protocols:

Preparation and Incubation

(1) Prepare the SIF and ensure it is at room temperature.

(2) Aseptically aliquot 5 mL of SIF into sterile tubes.

(3) Gently add 10 hydrogel beads to each tube. Designate tubes as Experimental (with bacteria) and Negative Control (blank beads).

(4) Place all tubes (caps loosened) into an anaerobic jar. Create an anaerobic atmosphere according to the manufacturer's instructions.

(5) Place the sealed anaerobic jar in a 37°C incubator for static culture.

Time-Point Sampling and Processing

(1) At each predetermined time point (Day 1, 3, and 7), remove the corresponding set of tubes from the incubator and anaerobic jar.

(2) Vortex each tube immediately at maximum speed for 10 seconds to ensure homogeneous suspension.

(3) Pipette 1 mL of the SIF incubation medium from each tube.

(4) Pass the 1 mL sample through a 0.22 µm sterile syringe filter into a new, sterile 1.5 mL microcentrifuge tube. Label the filtrate tubes clearly.

(5) Proceed directly to DNA extraction or store the filtrate at -80°C to prevent nucleic acid degradation.

qPCR Detection and Analysis

(1) Extract total DNA from 500 µL of each filtrate sample using the commercial DNA extraction kit, following the manufacturer's instructions. Elute the DNA in 50 µL of the provided elution buffer.

(2) Prepare a standard curve for absolute quantification by making a 10-fold serial dilution of the purified plasmid DNA standard (e.g., from 10⁸ copies/µL to 10¹ copies/µL).

(3) On ice, prepare the qPCR reaction mixtures in sterile PCR tubes or plates. For each 20 µL reaction:

\*10 µL of qPCR Master Mix

\*0.4 µL of Forward Primer (10 µM)

\*0.4 µL of Reverse Primer (10 µM)

\*2 µL of DNA template (from step (3).1) or standard

\*Nuclease-Free Water to a final volume of 20 µL

(4) Include appropriate controls in the qPCR run: No-Template Control (NTC, water instead of DNA) and a filtrate sample from the Negative Control group.

(5) Place the reaction plate in the qPCR instrument and run the following program:

\*Stage 1: 95°C for 5 minutes (Initial Denaturation)

\*Stage 2: 40 cycles of:

\*95°C for 15 seconds (Denaturation)

\*60°C for 1 minute (Annealing/Extension)

(6) After the run, use the qPCR software to analyze the data. Generate a standard curve from the dilution series and allow the software to calculate the absolute copy number concentration for each sample.

(7) Express the final results as "Target Gene Copies per mL of SIF". Perform statistical analysis to compare the gene copy numbers between the Experimental group and the Negative Control group at each time point.

5. Precautions:

(1) Maintain strict aseptic technique throughout the procedure to prevent microbial contamination. Use nuclease-free consumables to avoid nucleic acid degradation.

(2) The 0.22 µm filtration step is critical. It must be performed correctly to ensure that only leaked genetic material, not intact bacteria, is present in the filtrate for qPCR analysis.

(3) Always include the Negative Control (blank beads) and a No-Template Control (NTC) in the qPCR setup. This is essential for identifying any background signal or contamination that could lead to false positives.

(4) Vortex the samples thoroughly before filtration to ensure any leaked material is uniformly suspended in the SIF, providing a representative sample.

(5) If the filtrate is used directly as qPCR template without DNA extraction, a test for PCR inhibition should be performed.

References

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[5] Xiao, Y., Gu, Y., Qin, L., Chen, L., Chen, X., Cui, W., Li, F., Xiang, N., & He, X. (2021). Injectable thermosensitive hydrogel-based drug delivery system for local cancer therapy. Colloids and Surfaces B: Biointerfaces, 200, Article 111581.

[6] Pan C, Li J, Hou W, Lin S, Wang L, Pang Y, Wang Y, Liu J. Polymerization-Mediated Multifunctionalization of Living Cells for Enhanced Cell-Based Therapy. Adv Mater. 2021 Apr;33(13):e2007379. doi: 10.1002/adma.202007379. Epub 2021 Feb 25. PMID: 33629757.

[7]Sriamornsak P, Thirawong N, Korkerd K. Swelling, erosion and release behavior of alginate-based matrix tablets[J]. European Journal of Pharmaceutics and Biopharmaceutics, 2007, 66(3): 435-450.

[8] LIAO Junqi, HUANG Haining, CHEN Yinguang. CO2 promotes the conjunctive transfer of multiresistance genes by facilitating cellular contact and plasmid transfer [J]. Environment International, 2019, 129:333-342.

[9] ZHANG Ye,GU A Z,CEN Tianyu,et al. Subinhibitory concentrations of heavy metals facilitate the horizontal transfer of plasmid-mediated antibiotic resistance genes in water environment[J]. Environmental Pollution,2018,237:74-82.

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