Overview
In synthetic biology, precise and reproducible measurements are the foundation of meaningful design. Without reliable data, it becomes impossible to compare results, model systems, or improve constructs.
iGEM highlights measurement as one of the key criteria for scientific excellence, even including it as a Gold Medal criterion, underlining its importance for the scientific community.
Our team applies for the Best Measurement Award not only to validate our results, but also to contribute a robust, quantitative framework for characterizing our hydrogel system; specifically, its ability to contain genetically modified cells while providing a biocompatible environment that supports their proliferation and production of target molecules.
We aimed to go beyond simple "it works" observations by developing standardized, repeatable assays that, to our knowledge, have not been described in previous iGEM protocols or in existing literature. Our goal is to provide accessible and reliable methods that any team could use to evaluate both cell containment efficiency and cell viability within a hydrogel matrix, without facing the same struggle to find suitable protocols as we did.
Measurement Design
To quantitatively evaluate the performance of our hydrogel system, we designed two complementary assays focusing on:
Cell Containment Efficiency – to assess the hydrogel's ability to prevent bacterial escape over time.
Cell Viability Inside the Matrix – to determine whether the encapsulated bacteria remain alive and functional within the hydrogel environment.
Both measurements were performed using replicable, standardized methods that can be easily adapted by future iGEM teams working with encapsulation systems or living biomaterials.
Measurements Set Up
For these experiments, we used two E. coli strains available in our lab: W3110 ΔompT (the final strain used in our project) and W3110. Both strains had been used in previous experiments. We selected one motile and one non-motile strain to evaluate the containment capability of the hydrogel.
First, we performed a motility test to confirm the differences between the two strains as described in the Experiences section under "Hydrogel Encapsulation and Functionality - Motility Test of W3110 ΔompT compared to W3110 motile".
Once this difference was established, we developed our own protocols for hydrogel synthesis and made sure to maintain the same hydrogel exchange surface/medium volume ratio throughout our tests. After optimizing this protocol, we created additional procedures to assess bacterial escape and survival rates, analyzing the results by plotting escape curves and survival histograms.
We also used scanning electron microscopy (SEM) to examine the hydrogel matrix and the spatial distribution of the cells. Additionally, we performed fluorescence microscopy to confirm that the encapsulated cells were indeed our engineered strains and that they were able to produce molecules such as GFP.
1. Cell Containment Assay: Hydrogel Bacterial Escape Test
Objective
To measure whether bacteria remain confined within the hydrogel marbles and to quantify any potential release into the surrounding medium.
Principle
If the hydrogel matrix efficiently contains the bacteria, the turbidity (OD) of the surrounding medium should remain constant over time. Any increase in turbidity indicates bacterial leakage from the hydrogel into the surrounding medium.
Experimental Setup
Hydrogel marbles containing genetically modified E. coli were incubated in LB medium supplemented with antibiotic. 3 replicates were analyzed for each bacterial strain tested under two conditions:
Since we had previously recorded the typical OD increase over time for free E. coli culture without hydrogel, we were able to use it as a reference for bacterial growth kinetics. To ensure accurate interpretation, we also prepared a negative control containing only sterile medium and antibiotic, used both as a contamination control and as the OD blank.
Controls:
Positive control: Free bacteria (without hydrogel).
Negative control: Medium only (blank).
OD₆₀₀ measurements were taken directly using sterile starter tubes at defined time points (every 30 min up to 24 h).
One of the biggest challenges during method development was ensuring sterility throughout the OD measurements. Traditional spectrophotometers require non-sterile cuvettes, which would have compromised our samples, as we needed to re-incubate the same media after each reading. We initially tested alternative measurement strategies (see Hydrogel Bacterial Test 1 protocol carnet manip), but they were not sufficiently reliable (Figure A)
Figure A: 1st failed Hydrogel Bacterial Escape Test Results
Eventually, we overcame this limitation by using a cell density meter compatible with sterile starter tubes, which allowed us to measure OD under completely sterile conditions without transferring our samples or risking contamination.
Data Analysis
OD₆₀₀ values were plotted as a function of time to evaluate the kinetics of bacterial release from the hydrogel matrix. Mean values and standard deviations were calculated from at least three biological replicates to ensure reproducibility and statistical reliability.
A plateau or absence of OD increase indicated 100% bacterial containment within the hydrogel. We established that a slight OD increase within 24 h could be explained by bacteria initially present in the surrounding buffer solution used to transfer the hydrogel beads into the tubes.
To minimize this background signal, we optimized our protocol by introducing a three-step washing procedure. Following this adjustment, we considered that a minimal OD increase after 24 h did not necessarily indicate hydrogel failure, but rather reflected residual bacteria from the transfer buffer.
Conversely, an OD increase comparable to that observed in the positive control (free bacterial culture) suggested partial or total bacterial escape into the medium.
OD measurements were translated into bacterial escape curves plotted on excel or RStudio to visualize differences between both strains and experimental conditions.
Results and Reproducibility
All measurements were repeated independently several times to ensure consistency. The final protocol was optimized for clarity and reproducibility, allowing other iGEM teams to directly replicate the experiment without additional calibration steps.
Repetition provided valuable data, enabling the creation of a collective plot that integrates results from all tests to illustrate overall reproducibility (Figure B).
Figure B: Bacterial escape from alginate marbles monitored by OD₆₀₀ over 24 h (mean values from tests 3, 4, and 5).
By comparing these profiles, we were able to quantitatively assess the containment capacity of our hydrogel formulation and correlate its structure and composition with bacterial retention efficiency.
Escape curves revealed a clear difference between the two strains, with motile cells showing a higher propensity to move through the hydrogel compared to the non-motile strain. Nevertheless, the majority of encapsulated cells remained confined within the hydrogel throughout the tested time frames.
To Go Beyond
These results are promising and suggest that introducing motility-reducing mutations could be considered in future engineering cycles to further reduce bacterial escape for potential applications.
Further optimization will include comparing these results with alternative hydrogel formulations, as described in the Results – Future Work, section 3: Poly-DL-γ-glutamate (PGA) hydrogel for biocontainment
Yet, we are confident that our methods already provide a clear and standardized approach for testing hydrogel-based cell containment. We hope future iGEM teams will save time by using our methods and compare their results to ours through the provided results in the hydrogel measurement section.
2. Cell Viability Assay
Objective
To assess whether the hydrogel matrix provides a supportive environment that allows encapsulated bacteria to remain alive and metabolically active.
Principle
After specific incubation times, bacteria were recovered from the hydrogel marbles and plated on LB agar to determine colony-forming units (CFUs). The number of colonies reflects the survival rate of bacteria within the hydrogel.
Experimental Setup
Hydrogel marbles containing genetically modified E. coli were incubated in 1X PBS medium supplemented. 3 replicates were analyzed for each bacterial strain:
Hydrogel marbles containing bacteria were incubated at 37 °C using eppendorf tubes.
At defined time intervals, marbles were mechanically disrupted, and their contents were serially diluted and plated.
Colony counts were compared across time points to determine cell viability over time.
One of the main challenges during method development was ensuring the disruption of the marbles under sterility throughout the process. To maintain a consistent hydrogel-to-medium ratio across all tests, we used one marble per Eppendorf tube, which facilitated controlled disruption and ensured reproducible plating conditions. This setup allowed us to reliably recover the supernatant for colony counting, while keeping the experimental parameters consistent between replicates.
Data Analysis
CFU counts were used to calculate survival rates. The data were represented as mean ± standard deviation, enabling statistical comparison across replicates.
During our first attempt, we did not obtain the desired results, but after carefully reanalyzing our methods and optimizing our protocols, we successfully obtained meaningful results in a second 24-hour test. Following this success, we conducted a third test, extending measurements up to 72 hours to evaluate long-term viability.
Results and Reproducibility
During the 3rd test, measures were repeated independently with identical concentrations and conditions to ensure consistency. The final protocol was optimized for clarity and reproducibility, allowing other iGEM teams to directly replicate the experiment without additional calibration steps.
Repetition provided valuable data, enabling the creation of a collective plot that integrates results from all 3 tests to illustrate overall reproducibility (Figure C).
Figure C: Histogram of cell survival in 1.5% alginate hydrogel after 72 h (mean values from tests 1, 2, and 3).
Survival assays showed that encapsulation did not compromise cell viability. The survival histograms indicate that a substantial proportion of cells remained alive inside the matrix, demonstrating that the hydrogel provides a protective environment compatible with bacterial growth, even after 72 hours.
To Go Beyond
These results are promising and as no prior iGEM team had reported a standardized method for evaluating bacterial viability in hydrogels, this assay also constitutes a new protocol contribution to the iGEM community.
It offers a quantitative, reproducible approach to measure bacterial survival in 3D matrices.
Further optimization will include comparing these results with alternative hydrogel formulations, as described in the Results – Future Work, section 3: Poly-DL-γ-glutamate (PGA) hydrogel for biocontainment
We are confident that our methods already provide a clear and standardized approach for testing hydrogel-based cell viability. We hope future iGEM teams will save time by using our methods and compare their results to ours through the provided results in the hydrogel measurement section.
Conclusion
Our hydrogel measurement assays demonstrate that the 1.5% alginate hydrogel effectively contains genetically modified E. coli while providing a biocompatible environment that maintains high cell viability over extended periods (up to 72 hours).
The bacterial escape tests confirmed that most cells remained confined, with motile strains showing only minimal leakage. Meanwhile, the survival assays verified that encapsulation did not compromise bacterial growth, highlighting the hydrogel's protective properties.
By performing multiple independent replicates and optimizing our protocols for sterility, reproducibility, and clarity, we have established a robust framework that can be directly used by future iGEM teams. These methods not only ensure reliable, quantitative measurements but also contribute new, standardized protocols for assessing hydrogel-based containment and bacterial viability in synthetic biology projects.
In summary, this section showcases a repeatable, validated approach for evaluating both containment efficiency and cell survival; a critical step toward rigorous, reproducible synthetic biology research and a strong candidate for the Best Measurement award.
All Hydrogel Measurement Results
Motility test:
1st Hydrogel Synthesis test:
Bacterial Escape Test Results:
Bacterial Survival Test Results:
