Module 4: Hardware For Delivery & Characterization
The hardware part mainly includes the improvement of hydrogel carriers and the design of a turbidostat, providing support for the practical production of oral bacterial agents and the optimization of genetic circuits. Our hydrogel design aims to achieve core objectives including protecting probiotic EcN from gastric acid and digestive enzymes to ensure its viability upon intestinal arrival, and boosting intestinal adhesion through thiolation modification to promote long-term colonization. The turbidostat, an in vitro intestinal model that maintains a stable bacterial density, can be used to simulate colonization dynamics, thereby bridging our in vitro findings and facilitating future in vivo experiments.
Here we only present the results related to wet lab experiments with our designed hardware. For details on the hardware setup and construction process, please turn to our Hardware page.
Part 1: Hydrogel Microsphere Delivery System for EcN
1.Preparation and characterization of hydrogel microsphere delivery carrier with protective and release properties
Figure 1. Schematic diagram of our hydrogel microsphere delivery system.
(1)Preparation of hydrogel microspheres and controlled release assay
E. coli Nissle 1917 was cultured overnight at 37 °C in LB liquid medium containing the appropriate antibiotics. The bacterial cells were harvested and diluted to a concentration of approximately 1×109 CFU/mL. This cell suspension was then mixed uniformly with a approximately 1% (M/M) SA or CMC solution. The mixture was extruded dropwise using a syringe into 0.1M calcium chloride solution to form hydrogel microspheres via crosslinking over 10 minutes, subsequently encapsulated within enteric capsules to form the final oral delivery carriers.
For the release assay, samples were suspended in 4 mL of simulated gastric fluid (SGF) for 1 hour. Then, the SGF-treated microspheres were collected and resuspended in 10 mL of simulated intestinal fluid (SIF) for 7 h. Samples were taken hourly, washed and diluted with sterile PBS, and plated on LB agar plates. The plates were incubated at 37 °C for 24 hours before colony counting. Microspheres without the enteric coating were subjected to the same procedure but were not treated with SGF, instead being left to stand for 1 hour before transfer to SIF.
Taking hydrogel microspheres prepared with 1% sodium alginate and 0.1 M calcium chloride as an example, we evaluated the protective efficacy of the capsule and microspheres by sequentially immersing the samples in SGF followed by SIF. As shown in Figure 29A-C, the enteric capsules maintained their structural integrity without significant change after SGF treatment. However, they dissolved almost completely within ten minutes upon transfer to SIF at 37 ℃. The release curve demonstrated that alginate microspheres alone offered negligible resistance to SGF, whereas the capsules effectively protected the microspheres from the harsh gastric environment. Compared to unprotected microspheres, the capsule-protected microspheres exhibited a slower release of probiotics. After 7 hours in SIF, the release from the protected microspheres was only 54.2% of that from the control group, suggesting the release process may not have reached its plateau. We hypothesize that small peptide chains generated during gelatin degradation may engage in weak electrostatic interactions or hydrogen bonding with the carboxyl groups on the alginate microsphere surface, thereby altering the release kinetics.
Figure 2. Construction and release test of hydrogel microspheres within enteric capsules. A. Enteric capsules treated with SGF at 37 ℃ for 1h. B. Residue of enteric capsules after SIF treatment at 37 ℃ for 10min. C. Dissolution of capsules in SIF. D. Effect of enteric capsule encapsulation on the release profile of hydrogel microspheres. E. Fully swollen hydrogel microspheres.
(2)Controlled Release of Hydrogel Microspheres
To achieve targeted delivery, it is crucial to control the hydrogel's release profile so that the majority of probiotics are released within a specific window period. We investigated this by varying the concentrations of sodium alginate (SA) and the crosslinking agent (Ca2+) to determine the optimal formulation.
The results showed negligible bacterial release within the first 4 hours, with most probiotics retained within the microspheres. After 4 hours, we observed a rapid and substantial release of probiotics into SIF, indicating the microspheres' potential to achieve precise delivery to specific regions of the intestinal tract. According to our literature review (see our Model page for details), the release profile and swelling ratio of the hydrogel can be theoretically tuned by adjusting the shrinkage affinity and the cross-link density, which are influenced by the reagent concentrations. However, varying the concentrations of SA or Ca2+ did not yield a significant difference in the controlled release behavior. This suggests that these particular concentration adjustments may not substantially alter the microporous density of the hydrogel, which governs the diffusion rate.
An intriguing finding was that the cumulative number of probiotics released after 7 hours in SIF exceeded the initial amount loaded into the hydrogel during preparation. This indicates that the microspheres provide a favorable microenvironment that can support subsequent bacterial growth and proliferation. As shown in Figure 30C and D, while most bacteria left the hydrogel via diffusion and swelling effects after 7h in SIF, a small fraction remained trapped within the gel pores. These trapped bacteria likely utilized diffused nutrients for proliferation, with their progeny gradually releasing into the surrounding fluid, leading to the observed surplus. In practical applications, this phenomenon could be advantageous, helping to retain a subpopulation of engineered EcN within the microspheres for sustained colonization and continuous GLP-1 synthesis.
Figure 3. Characterization of the controlled release properties of hydrogel microspheres. A. Viability and accumulation of E. coli released from microspheres. B. Release profiles of probiotic carriers with different cross-linking degrees. C. Microstructure of fully swollen hydrogel microspheres under light microscopy (1000x). D. Microstructure of hydrogel microspheres after 7h of release in SIF under light microscopy (1000x).
(3)Summary
Our findings highlights the potential of hydrogel microspheres-enteric capsules combination strategy to create a protective "Noah's Ark" for probiotics, supporting not just delivery but also their proliferation and colonization. While literature suggests advanced bilayer designs or precise size control for enhanced targeting, we found enteric capsules to be a pragmatically superior solution due to their simpler engineering, reliability, and public acceptance. Future work should focus on optimizing microsphere parameters like porosity and size to establish a quantitative model linking carrier structure to probiotic function.
2.Characterization of adhesion property of thiol-modified hydrogel
After thiolation modification of carboxymethyl cellulose (CMC-SH), it can form strong disulfide bonds with cysteine, thiol-containing proteins, and other components on the intestinal mucosa and bacterial surface, thus significantly enhancing its adhesiveness. We used CMC-SH to roughly determine its adhesive properties through a simple running water rinsing experiment (see our experimental protocol for details), providing support for the long-term colonization of engineered probiotics.
The EcN strain used here had been transformed with dual plasmids carrying ampicillin (Amp) and streptomycin (Str) resistance genes. Therefore, we spread the strain on LB agar plates containing double antibiotics (AmpR and StrR). We first streaked the back of the plate to divide it into two equal parts, and then spread a thin layer of prepared CMC hydrogel or CMC-SH hydrogel on one side, while leaving the other side untreated. After the hydrogel solidified, we streaked both the hydrogel-coated area and the uncoated blank agar area with an inoculating loop for inoculation, placed the plates at 37 °C for inverted incubation overnight until the bacterial colonies grow into a clear pattern.
Through a simple running water rinsing experiment, we observed that in both types of plates, bacteria in the agar areas without hydrogel coating were easily rinsed off. In contrast, the CMC-SH coating and the bacteria adhering to its surface were much harder to be rinsed off compared with the CMC coating, showing little difference from the unwashed ones. From this, we preliminarily concluded that thiol-modified CMC is capable of enhancing its adhesive capacity to bacterial cells, even for bacteria not obviously embedded, which is conducive to the long-term colonization of engineered bacteria in the intestine.
Figure 4. Comparison of adhesion performance of CMC and CMC-SH hydrogel coatings on LB agar plates for EcN based on the running water rinsing experiment
Adhesion and Rinse Experiment Operation Video of CMC-SH Hydrogel Experimental Group and Blank Control Group
Part 2: In Vitro Turbidostat for Circuit Characterization
Since shake flasks, culture tubes, and other vessels in the laboratory are all batch culture induction systems that require simultaneous addition of inducers at time zero, and the initial OD600 value of the bacterial strain is only about 0.02, there is a certain asynchrony between bacterial growth and gene circuit expression. Consequently, the measured induction time curves often differ significantly from in vivo induction expression. Therefore, we have made corresponding improvements using a turbidostat device.
A turbidostat is originally used for microbial culture and state monitoring. Here, we mainly simulate its characteristics of constant temperature, stirring, and inlet-outlet to control the constancy of bacterial density in the culture system, thereby real-time simulating the induction response behavior of bacterial gene circuits and providing preliminary data basis for the induction scheme in in vivo contexts.
1.Construction of in vitro turbidostat
We constructed a simple in vitro turbidostat model using devices including a feeding device, reactor, peristaltic pumps, and a thermostatic magnetic stirrer. The three-necked flask was entirely placed in a water bath for constant-temperature cultivation at 37 ℃. A peristaltic pump on the left side continuously pumps in fresh LB medium, with its flow rate dynamically regulated based on the OD600 value of the bacterial culture in the flask. Another peristaltic pump on the right side continuously aspirates the bacterial culture. The outflow of bacterial culture is regulated by controlling the insertion length of the catheter, maintaining a roughly constant volume of bacterial culture. A collection tube is connected to the outlet to collect the bacterial culture in real time, which is used to determine the OD600 and the Fluo/OD600 ratio of the bacterial culture.
We first inoculated a bacterial culture with an initial OD600 of 0.02 into the three-necked flask holding 50 mL of LB broth, and incubated it overnight for preculture. Subsequently, we sampled from the collection tube at fixed time intervals to determine the OD600 value, and adjusted the flow rates of the inlet and outlet liquids based on the measured results to judge whether the system had reached a turbidostatic state. Furthermore, we controlled the OD600 value to fluctuate around 1.4 with reference to relevant literature. Ultimately, we successfully achieved the maintenance of a turbidostatic state for nearly 5 hours.
Figure 5. Construction of a simple in vitro turbidostat. A. Physical model of the simple turbidostat. B. OD600 measurement results of the turbidostat showing maintenance of turbidostatic states after 4h testing.
2.Characterization of genetic circuits in the turbidostat
After adjusting the inlet and outlet flow rate parameters to stabilize the OD600 value, we conducted induction tests on E. coli harboring the dual plasmids of the AND-gate circuit. When the system reached a turbidostatic state, an appropriate amount of PCA and sodium cholate was simultaneously injected into the flask containing the bacterial suspension, reaching final concentrations of 400 μM and 25 μM, respectively. Taking the moment of inducer injection as time zero, samples were taken every half hour to determine the unit fluorescence intensity and analyze the trend of fluorescence changes with induction time.
The experimental results showed that the Flou/OD600 values in the above experiment exhibited a trend of first increasing and then decreasing, reaching a peak at approximately 3 hours. This indicates that our AND-gate sensing engineered bacteria can quickly respond to induction by the green tea metabolite PCA in the “simulated intestine” and synthesize downstream products at around 3 hours. In practice, induction in the human intestine may have a shorter response time, which means that the oral bacterial agent can allow users to take green tea 3-4 hours before meals for pre-synthesis of GLP-1, providing a reference for in vivo experiments and industrial applications.
Figure 6. Induced fluorescence measurement results of strains harboring the AND-gate circuit plasmids in the turbidostat for 4 hours.
3.Summary
The simple in vitro turbidostat serves as a supplementary tool for in vivo experiments under laboratory conditions. By simulating the dynamic stability of bacterial density in the intestine, it provides a basis for optimizing the induction time and dosage of inducers in practical applications. However, during the actual measurement process, we only detected the response time of the AND-gate circuit and did not characterize the relevant properties of subsequent plasmids. Furthermore, the experiment only maintained the turbidostatic state of a single bacterial strain, which still differs from the real intestinal environment. In addition, since PCA and BS were added in a single dose, their concentrations changed with the inflow and outflow of the culture medium, thereby affecting the final fluorescent expression. In subsequent studies, it is necessary to evaluate the existing simplified assumptions and the rationality of the results based on the above issues, and further optimize the turbidostatic conditions to effectively improve the reliability of the conclusions.
Conclusions
The hydrogel delivery system and the in vitro turbidostat reactor simulation are our critical tools for developing engineered probiotic products. These experiments have provided us with deeper insights into gut microbiota complexity and oral drug delivery, enabling us to integrate all modules into a cohesive platform. We anticipate that future advancements in hardware tools that better mimic the human intestinal microenvironment will further enhance simulation systems for engineered bacteria, specifically for their delivery and colonization, the production and secretion of therapeutic agents, and the monitoring of substrate metabolism.