iGEM 2025 — Team latvia-riga

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{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
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## What Should this Page Contain?

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* Record all activities in chronological order, with each entry clearly dated.
* Provide sufficient detail for each activity, including:

+ Purpose of the activity.
+ Methods and procedures used.
+ Results obtained.
+ Observations and notes.

* Document all experiments, protocols, data analysis, meetings, brainstorming sessions, and other project-related activities.
* Include photos, videos, and diagrams to illustrate your progress.
* Indicate which team members participated in each activity.

## Inspirations

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* [2024 EPFL](https://2024.igem.wiki/epfl/notebook)
* [2024 JU-Krakow](https://2024.igem.wiki/ju-krakow/notebook)

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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## Colony PCR

Design

As part of our wet lab process to confirm successful cloning of our constructs and transformation into bacterial cells, we needed to perform colony PCR. In theory, this is a straightforward process: a PCR reaction mix is prepared according to protocol, but instead of template DNA, a tiny piece of a transformant colony—which should contain the successfully cloned gene fragment—is used. After PCR and gel electrophoresis, we expect to see a band of amplified DNA at the correct length.

Build

After cloning and transforming our plasmids into bacterial cells, the cells were incubated for up to 48 hours. Five colonies from each transformant were randomly selected to test for successful cloning. Colony PCR was performed according to the protocol, and the products were run on an agarose gel.

Test

The electrophoresis gel was visualised and analysed.

Learn

On the first colony PCR gel, we observed a large, glowing cloud in the region where we expected the amplicon DNA band. Even if the amplicon band was present, the glowing cloud obscured it. We hypothesised that this cloud might be cellular debris or other contaminants left over from cell lysis during the PCR. To address this, we theorised that centrifuging the PCR tubes could pellet any insoluble debris.

![First colony PCR: large glowing cloud observed in each well; image cropped to highlight the issue.](https://static.igem.wiki/teams/5728/engineering/efig1.webp)

*Figure 1. First colony PCR, a large, glowing cloud can be observed for each well. Picture is cropped to better visualise the problem.*

Design

One challenge was that we lacked a centrifuge capable of spinning 0.2 mL PCR tubes at the required speed. To solve this, we designed 3D-printed adapters that would fit into a standard centrifuge carrier for 2 mL tubes.

Build

The adapters were 3D-printed. Colony PCR was repeated, and this time, before gel electrophoresis, the tubes were centrifuged to sediment any debris. The supernatant was then loaded onto the agarose gel, and electrophoresis was performed.

Test

The electrophoresis gel was visualised and analysed.

Learn

Unfortunately, the glowing cloud persisted, indicating that centrifugation did not resolve the issue. After consulting our supervisor, it was suggested to add RNase to the PCR mix to degrade any RNA present.

![First colony PCR: large glowing cloud observed in each well; image cropped to highlight the issue.](https://static.igem.wiki/teams/5728/engineering/efig2.webp)

*Figure 2. Expression at 37 degrees 4 h VS 20 degrees overnight. BCBI - BL21(DE3) cells before induction; WCL - whole cell lysate; LS - lysate supernatant; CD - cell debris.*

Design

A revised colony PCR protocol was designed, incorporating additional magnesium dichloride and RNase.

Build

The new protocol was tested on the same colonies, but on a smaller scale, to evaluate the effect of adding RNase. PCR was performed, and samples were loaded onto the gel—this time without centrifugation.

Test

The electrophoresis gel was visualised and analysed.

Learn

Analysis revealed that the "cloud of doom" was indeed RNA, as the addition of RNase eliminated the cloud. The gel was clear, and the DNA amplicon bands were clearly visible. Although, further on different primers were also used, which produced a longer amplicon, and wouldnt have been in the RNA cloud anyways.

![First colony PCR: large glowing cloud observed in each well; image cropped to highlight the issue.](https://static.igem.wiki/teams/5728/engineering/efig3.webp)

*Figure 3. Colony PCR when RNase was added as a part of the PCR mixture. Lack of a glowing cloud can be observed, and clear amplicons are visible.*

## Hydrogels

Design

For hydrogel preparation, we decided to follow a protocol described in F.song et al., 2009 [1]. This protocol specified a final casein concentration of 8% w/w, with three genipin concentrations: 2.5 mM, 5 mM, and 10 mM. It also recommended crosslinking at 50°C to accelerate the process.

Build

We followed the protocol, but many details were ambiguous (e.g., where to dissolve genipin), and several steps required assumptions. Despite this, we prepared the hydrogel mixture, pipetted it into HPLC vials, and incubated it at 50°C for the suggested duration.

Test

After incubation, we inverted the vials to check for hydrogel formation. Unfortunately, the mixture remained as runny as before, with no increase in viscosity—though it had turned dark blue.

Learn

No hydrogel formed, so no further tests could be performed. However, the color change from transparent to dark blue suggested a reaction between genipin and casein proteins had occurred. The exact reason for the lack of gelation remained unclear.

![Figure 4. Hydrogel mixture before incubation.](https://static.igem.wiki/teams/5728/engineering/efig4.webp)

*Figure 4. Hydrogel mixture before incubation.*

![Hydrogel mixture after incubation. Solutions were still liquid, but a clear colour change was observed.](https://static.igem.wiki/teams/5728/engineering/efig5.webp)

*Figure 5. Hydrogel mixture after incubation. Solutions were still
 liquid, but a clear colour change was observed.*

Design

We hypothesized that the crosslinking time might have been insufficient, so we decided to repeat the experiment with an extended incubation period of 24 hours.

Build

The hydrogel mixture was prepared as before, with the only change being the increased incubation time.

Test

After 24 hours, we again inverted the vials to test for hydrogel formation. The mixture was still liquid, though the same dark blue color change was observed.

Learn

The test for hydrogel formation failed once more. The mixture remained non-viscous, and no conclusions could be drawn beyond the color change. We suspected that the protocol’s lack of specific details might have contributed to the failure.

Design

Next, we switched to a different protocol N.F.N. Silva et al., 2014 [2]. This time, we dissolved a lower concentration of casein in HEPES buffer with added calcium chloride, adjusted the pH to 7.10, and prepared genipin in HEPES buffer with absolute ethanol to a concentration of 200 mM. The final concentrations in the hydrogel mixture were set to 25 g/L casein and 5 mM, 10 mM, or 20 mM genipin.

Build

We followed the new protocol precisely, without needing to guess any steps or values. The hydrogel mixture was prepared in HPLC vials and incubated in a 50°C water bath for 24 hours.

Test

After incubation, we performed the vial inversion test again. Disappointingly, the mixture remained liquid, despite the same transparent-to-dark-blue color change.

Learn

From these tests, we concluded that forming hydrogels from casein and genipin is more complex than initially anticipated. After further literature review, we suspect that sodium caseinate may not be suitable for hydrogel formation using genipin. Instead, assembled casein micelles or differently processed casein proteins might be required.

![Figure 6. Hydrogel mixture before incubation.](https://static.igem.wiki/teams/5728/engineering/efig6.webp)

*Figure 6A. Hydrogel mixture before incubation.*

![Hydrogel mixture after incubation. Solutions were still liquid, but a clear colour change was observed.](https://static.igem.wiki/teams/5728/engineering/efig7.webp)

*Figure 6B. Hydrogel mixture after incubation.
 Solutions were still liquid, but a clear colour change was observed.*

**Additional Test for Crosslinking**

As an additional test, we performed SDS-PAGE under non-reducing conditions to assess potential crosslinking. If crosslinking had occurred, we expected to see an increase in molecular weight. However, the results were inconclusive: while some staining was observed, it remained at the start of the separating gel. This could indicate extensive crosslinking, preventing the proteins from entering the gel—but such a conclusion is unreliable.

![SDS-PAGE performed under non-reducing conditions](https://static.igem.wiki/teams/5728/engineering/efig8.webp)

*Figure 7. SDS-PAGE performed under non-reducing conditions.*

**Next Steps**

Further research is needed, including more extensive literature review, potential re-micellisation of casein, or alternative procedures.

## Propolis antimicrobial property tests on *E. coli* and *S. cerevisiae*

Design

For antimicrobial testing, propolis was extracted using PEG 400 to avoid ethanol-related growth inhibition. The concentration of phenolic compounds was assumed to be 10.7 ± 1.2 mg/mL, based on a publication from which the protocol for propolis extract was sourced L. Kubiliene et al., 2015 [3]. Dual-layer agar plates with agar well diffusion methods were employed, following the same publication.

Build

We prepared the propolis extract and inoculated plates with E. coli and S. cerevisiae (separately). Four wells were punched into the agar layer, and 100 µL, 150 µL, and 200 µL of propolis extract were pipetted into the wells on separate plates. The plates were incubated for 48 hours.

Test

After 48 hours of incubation, the plates were scanned and analysed digitally.

Learn

In the scanned images, no inhibitory effect from propolis was observed. While observing the plates with the naked eye, we noticed a slight reduction in microbial growth, but not enough for any meaningful analysis. We concluded that this method might not be suitable for our needs, especially since we were unable to precisely determine the phenolic content of our propolis extract due to a lack of proper tools. Additionally, we theorised that liquid extract diffusion from agar wells might be too slow because the plates were freshly poured.

![Scan of dual-layer agar plates with 4 wells for propolis diffusion. ](https://static.igem.wiki/teams/5728/engineering/efig-9.webp)

*Figure 8. Scan of dual-layer agar plates with 4 wells for propolis diffusion. Top right and bottom right: plates inoculated with E.coli. Top left and bottom left: plates inoculated with S.cerevisiae.*

Design

Based on the previous iteration, we decided to repeat the experiment using plates that had been left to dry slightly in an aseptic environment, allowing the liquid propolis extract to diffuse better and faster. A positive control with sterile 20% PEG 400 solution—the same solution used for preparing the propolis extract—was also added.

Build

The same protocols were followed, incorporating the changes mentioned above.

Test

After incubation, the plates were scanned for digital analysis.

Learn

Unfortunately, the results did not differ from the previous experiment. No visible inhibition of microbial growth was observed around the agar wells containing propolis extract or the 20% PEG 400 solution. As before, slight growth reduction was noticed with the naked eye, but it was insufficient for analysis. We decided to try another inoculation method using a colony assay stamp we had designed and 3D-printed. Instead of the dual-layer agar method, we would use agar plates with colonies stamped in a specific arrangement, featuring a central well for the propolis extract.

Design

As mentioned, we used the 3D-printed colony assay stamps to assess the antimicrobial effect of propolis. Other methods remained unchanged.

Build

The colony assay stamps were prepared for use. Plates that had been left to dry in an aseptic environment were inoculated with the respective microorganism using the stamps. A central well was punched out and filled with propolis extract. The plates were incubated for 48 hours.

Test

After 48 hours of incubation, the plates were observed and scanned for documentation.

Learn

This time, smaller colonies were observed closer to the well containing propolis extract, with larger colonies appearing farther away, especially at the edges of the colony assay. While this suggested a potential antimicrobial effect, we also considered that nutrient competition among colonies might have contributed to the reduced growth of inner colonies. For further testing, we decided to use 96-well plate assays to improve repeatability and data reliability.

![Colony assay with a central well for propolis diffusion. Test on S.cerevisiae.](https://static.igem.wiki/teams/5728/engineering/efig10.webp)

*Figure 9. Colony assay with a central well for propolis diffusion.
 Test on S.cerevisiae.*

![Colony assay with a central well for propolis diffusion. Test on E.coli.](https://static.igem.wiki/teams/5728/engineering/efig11.webp)

*Figure 10. Colony assay with a central well for propolis diffusion.
 Test on E.coli.*

Design

The 96-well plate assay was designed to test the antimicrobial effect of propolis at different concentrations. This approach would allow us to quantify the results and determine the amount of extract needed for an antimicrobial effect in liquid culture. To account for any inhibitory effects from the 20% PEG 400 solution, control repetitions were included using the PEG solution at the same volumes as the propolis extract. A negative control with only media (and the same volume of extract solvent) was also added.

Build

The 96-well plates were prepared and filled with the respective propolis extract concentrations. After inoculation, the plates were placed in a plate reader.

Test

Optical density data over time was measured and analysed using the plate reader.

Learn

Analysis revealed that higher concentrations of propolis extract correlated with increased inhibition of bacterial growth, confirming the presence of antimicrobial compounds in our extract. However, the inhibitory effect was weaker than expected. Increasing the extract volume was considered, but its opacity would interfere with optical density measurements, compromising data reliability. To address this, we proposed producing stronger extracts by using more propolis.

|  |  |  |
| --- | --- | --- |
| ![96-well plate assay for testing propolis antimicrobial properties.](https://static.igem.wiki/teams/5728/engineering/efig12.webp)  *Figure 11. 96-well plate assay for testing propolis antimicrobial properties. S.cerevisiae in the top plate, E.coli in the bottom plate. Assays pictured after the experiment.* | ![Growth curves of S.cerevisiae in presence of increasing concentrations of Propolis and PEG solution.](https://static.igem.wiki/teams/5728/engineering/efig13-1.webp)  *Figure 12. Growth curves of S.cerevisiae in presence of increasing concentrations of Propolis and PEG solution.* | ![Growth curves of E.coli in presence of increasing concentrations of Propolis and PEG solution](https://static.igem.wiki/teams/5728/engineering/efig14-1.webp)  *Figure 13. Growth curves of E.coli in presence of increasing concentrations of Propolis and PEG solution.* |

## Protein expression

Design

To produce our proteins of interest, an E. coli expression system was selected due to its typically high protein yield, short expression time, and low production cost. Moreover, protein production in E. coli is easily scalable, allowing to express the proteins in analytical amounts for the evaluation of various expression conditions to identify the optimal parameters and later scale up to obtain higher amounts of protein of interest for further applications. For protein production the canonical BL21(DE3) was chosen as a reliable option for recombinant protein expression.

Build

BL21(DE3) competent cells were transformed with plasmid constructs containing genes coding for proteins of interest. For protein expression, transformed cells were cultivated at 37°C, shaking at 180 rpm, induced with 1 mM final concentration of IPTG at OD600 0.6 - 0.9 and cultivated in the same conditions for 4 h afterwards.

Test

Small-scale protein expression and solubility tests were performed to evaluate protein expression efficacy in described conditions. Results were analysed by SDS-PAGE. We managed to successfully express the recombinant α-S1-casein containing 6xHisTag and TEV-site.

Learn

We learnt that our α-S1-casein is being expressed by the engineered E. coli, however, it is highly insoluble. At this point we started to look for optimisation possibilities for protein expression protocols to enhance soluble protein expression.

![Analysis of α-S1-casein production efficiency and solubility by SDS-PAGE. Protein was expressed in E. coli BL21(DE3) at 37 °C for 4 hours. ](https://static.igem.wiki/teams/5728/engineering/efig15.webp)

*Figure 14. Analysis of α-S1-casein production efficiency and solubility by SDS-PAGE. Protein was expressed in E. coli BL21(DE3) at 37 °C for 4 hours.*

Design

During research we found out that lowering the expression temperature and final IPTG concentration for induction promotes slower protein expression, which often results in higher yields and improved solubility of the proteins produced. For this reason it was decided to apply a protein production optimisation protocol for α-S1-casein expression in hopes of improving soluble protein expression and possibly obtaining higher protein yields

Build

BL21(DE3) cells that were previously transformed with our plasmid constructs were cultivated at 37 °C, shaking at 180 rpm until the OD600 reached 0.4 - 0.5, after which the temperature was lowered to 20°C, the cell culture was cooled, and protein expression was induced with 2x lower final concentration of IPTG at OD600 0.6 - 0.9. Protein production was carried out overnight.

Test

Protein production efficiency and solubility were tested once again to evaluate the effect of protocol modifications. The results were analyzed by SDS-PAGE.

Learn

We learnt that lowering the expression temperature indeed optimises protein production and yields a higher overall protein content. However, it did not contribute much to solubilising the recombinant α-S1-casein.

![Analysis of α-S1-casein production efficiency and solubility by SDS-PAGE.](https://static.igem.wiki/teams/5728/engineering/efig16.webp)

*Figure 15. Analysis of α-S1-casein production efficiency and solubility by SDS-PAGE. Protein was expressed in E. coli BL21(DE3) at 20 °C overnight.*

Design

Since the bovine α-S1-casein produced was mostly insoluble with a small soluble protein fraction in relation to the overall amount of protein produced, we decided to use a different strain of E. coli to see if we can manage to increase the fraction of soluble proteins produced by the E. coli. For that we used an E. coli strain, Rosetta 2, which is a derivative of BL21 (DE3) optimised for expression of eukaryotic proteins.

Build

A new successful transfection was performed, using the same α-S1-casein gene-containing plasmid construct and competent Rosetta 2 E. coli cells. Since earlier experiments clearly showed the advantage of expressing proteins in E. coli at lower temperature overnight, protein production experiments in the Rosetta 2 strain were carried out in the same conditions – with induction of 0.5 mM final concentration of IPTG and protein expression overnight at 20°C with shaking at 180 rpm.

Test

Protein production efficiency and solubility after expression in the E. coli Rosetta 2 strain were tested to evaluate if using an alternative strain improves the solubility of the protein and offers comparatively better yields of soluble proteins. Analysis was done by SDS-PAGE.

Learn

We found that using Rosetta 2 cells for the expression of α-S1-casein offers a similar overall protein yield compared to the BL21(DE3) system in the same expression conditions, indicating that this strain is also suitable for producing our protein of interest. However, it did not significantly contribute to solubilising the α-S1-casein, as no notable improvement was observed in the amount of soluble protein obtained. Protein expression is happening at a high level, although additional optimisation steps are required to increase the solubility. One of the possible approaches would be to introduce solubility tags and test if that has any positive impact on the outcome of protein expression

![Comparative analysis of α-S1-casein production efficiency and solubility by SDS-PAGE in BL21 (DE3) strain at 37 °C, in BL21(DE3) strain at 20 °C and Rosetta 2 strain at 20 °C.](https://static.igem.wiki/teams/5728/engineering/efig17.webp)

*Figure 16. Comparative analysis of α-S1-casein production efficiency
 and solubility by SDS-PAGE in BL21 (DE3) strain at 37 °C, in BL21(DE3) strain at 20 °C and Rosetta 2 strain at 20 °C. Protein expression at 20 °C significantly improves the production yield, however no notable improvements are detectable for the increase of soluble protein fraction.*

References

[1] F. Song, L.-M. Zhang, C. Yang, and L. Yan, “Genipin-crosslinked casein hydrogels for controlled drug delivery,” International Journal of Pharmaceutics, vol. 373, no. 1–2, pp. 41–47, Feb. 2009, doi: 10.1016/j.ijpharm.2009.02.005.

[2] N. F. N. Silva, A. Saint-Jalmes, A. F. De Carvalho, and F. Gaucheron, “Development of Casein Microgels from Cross-Linking of Casein Micelles by Genipin,” Langmuir, vol. 30, no. 34, pp. 10167–10175, Aug. 2014, doi: 10.1021/la502274b.

[3] L. Kubiliene et al., “Alternative preparation of propolis extracts: comparison of their composition and biological activities,” BMC Complementary and Alternative Medicine, vol. 15, no. 1, May 2015, doi: 10.1186/s12906-015-0677-5.

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{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
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**Results: successful expression and purification of a target protein - bovine origin α-S1-casein**

System Selection and Initial Expression

To produce our proteins of interest, an E. coli expression system was selected due to its high protein yield, rapid expression time, and cost-effectiveness. The BL21(DE3) strain, a standard host for recombinant protein production, was chosen for its reliability and scalability—allowing initial optimization in small-scale cultures before scaling up for larger applications. Transformed BL21(DE3) cells were cultivated at 37°C with shaking at 180 rpm, and protein expression was induced with 1 mM IPTG at an OD600 of 0.6, followed by a 4-hour incubation under the same conditions.

Small-scale expression and solubility tests, analyzed by SDS-PAGE, confirmed that the target protein was successfully expressed. However, the majority of the protein was found in the insoluble fraction, indicating the formation of inclusion bodies. This outcome prompted the need to optimize expression conditions to enhance soluble protein yield.

Optimization of Expression Conditions

Based on literature and empirical evidence, we hypothesized that reducing the expression temperature and IPTG concentration could slow protein synthesis, potentially improving solubility. The protocol was adjusted as follows: cells were grown at 37°C to an OD600 of 0.4–0.5, after which the temperature was lowered to 20°C, and induction was performed with half the original IPTG concentration (0.5 mM). Protein production was then carried out overnight to allow for slower, more controlled synthesis.

Evaluation of Optimized Conditions

Solubility and expression efficiency were reassessed by SDS-PAGE (Fig.1). While the modified protocol increased overall protein yield, likely due to prolonged expression at a lower temperature, the target protein - α-S1-casein - remained predominantly insoluble.

Additional Optimization and Evaluations

Even after primary expression condition optimization, bovine α-S1-casein produced was primarily insoluble with a relatively small fraction of soluble protein. To address this, we used the Rosetta 2 strain of E. coli, which is optimized for eukaryotic protein expression. We tested protein production efficiency and solubility using SDS-PAGE analysis. The results indicated that while Rosetta 2 provided a similar overall protein yield compared to the BL21(DE3) strain, it did not significantly improve the solubility of α-S1-casein. These results suggest that additional strategies, such as co-expression with chaperones, use of solubility-enhancing tags, or alternative host strains, may be required to achieve soluble expression of α-S1- casein in E. coli [1].

![Results](https://static.igem.wiki/teams/5728/illustrations/result1.avif)

*Figure 1. Comparative analysis of protein production efficiency and solubility in different expression systems and expression conditions. Lanes 1–3: BL21 (DE3) strain at 37°C—whole cell lysate, lysate supernatant, and cell debris, respectively. Lanes 4–6: BL21 (DE3) strain at 20°C—whole cell lysate, lysate supernatant, and cell debris, respectively. Lane 7: well overflow. Lanes 8–10: Rosetta 2 strain at 20°C—whole cell lysate, lysate supernatant, and cell debris, respectively. Lane 11: Rosetta 2 whole cell lysate before induction.*

Since the bovine α-S1- casein protein exhibited partial solubility, it was sufficient to enable the purification of the soluble fraction using HisTrap FF Ni²⁺ affinity chromatography. This finding is significant as it indicates that, despite the partial insolubility, a functional portion of the protein can be obtained for further study. The successful purification of the α-S1- casein protein using HisTrap Ni²⁺ affinity chromatography was confirmed by SDS-PAGE analysis of the eluate fractions, which revealed that functional 6xHisTag has been successfully integrated into the protein amino acid sequence (fig. 2). The presence of the 6xHisTag is crucial as it allows for the specific binding and elution of the protein from the affinity column, facilitating its purification. The efficiency of the purification process was demonstrated by the clear presence of the α-S1- casein protein in the eluate fractions, with minimal contamination from other cellular proteins. This successful purification opens the door for possible further applications of the α-S1- casein in the overall context of our project.

![Results](https://static.igem.wiki/teams/5728/illustrations/result2.avif)

*Figure 2. Results of expressed recombinant bovine origin α-S1-casein purification by Ni2+ affinity chromatography with HisTrap FF column. Lane 1: Rosetta 2 cells before induction. Lane 2: Clarified cell lysate supernatant prior to HisTrap FF column application. Lane 3: Flow-through lysate supernatant after loading on HisTrap FF column. Lane 4: Column wash fraction. Lane 5: Wash fraction with binding buffer containing 20 mM imidazole. Lanes 6–10: Elution fractions using 300 mM imidazole buffer. Lane 11: Cell debris.*

Conclusion and Next Steps

The optimization of temperature and inducer concentration improved total protein production but did not resolve the solubility issue for α-casein. Future efforts will focus on exploring alternative expression systems, such as Saccharomyces cerevisiae or Kluveromyces marxianus, or employing fusion tags (e.g., MBP, GST) to enhance solubility. Additionally, refining lysis and refolding protocols could be considered to recover functional protein from inclusion bodies.

Results: Attempted Preparation of Casein-Genipin Hydrogels

The objective of this study was to prepare hydrogels by cross-linking casein proteins with genipin, following established protocols and optimizing reaction conditions. Despite systematic experimentation, hydrogel formation was not achieved, though chemical interaction between genipin and casein was confirmed by a characteristic color change.

Experimental Approach and Observations

Initial Protocol

The first protocol, adapted from [doi:10.1016/j.ijpharm.2009.02.005], specified a final casein concentration of 8% (w/w) and genipin concentrations of 2.5 mM, 5 mM, and 10 mM, with crosslinking at 50°C. After preparing the hydrogel mixture and incubating for the suggested duration, vial inversion tests revealed no increase in viscosity; the mixture remained liquid, albeit with a dark blue color indicative of genipin-casein interaction. Extending the incubation to 24 hours did not yield a hydrogel, suggesting that either the crosslinking time or other protocol details (e.g., solvent choice, mixing order) were insufficient for gelation.

Revised Protocol

A second protocol was implemented, using a lower casein concentration (25 g/L) dissolved in HEPES buffer with calcium chloride (pH 7.10) and genipin prepared in HEPES buffer with absolute ethanol (final genipin concentrations: 5 mM, 10 mM, 20 mM). After 24 hours at 50°C, the mixture again failed to gel, despite the persistent blue color change.

Crosslinking Assessment

SDS-PAGE under non-reducing conditions was performed to evaluate potential crosslinking. While some protein staining was observed at the gel origin, likely due to high-molecular-weight aggregates, the results were inconclusive. Such staining could indicate extensive crosslinking preventing protein migration, but this interpretation remains speculative without further evidence.

Discussion

The consistent color change from transparent to dark blue confirmed that genipin reacted with casein primary amine groups, as reported in the literature. However, the absence of gelation suggests that sodium caseinate—used in both protocols—may not be suitable for hydrogel formation under these conditions. Casein s native structure, typically present as micelles in milk, is disrupted in sodium caseinate, potentially hindering the formation of a stable three-dimensional network required for hydrogel formation. Successful hydrogel preparation with genipin often relies on intact protein structures or assembled micelles, which provide the necessary physical interactions for gelatination.

Conclusion

Propolis extract demonstrated antimicrobial activity in liquid culture, but its efficacy was weaker than anticipated. The 96-well plate assay proved more reliable for quantifying growth inhibition, though extract opacity constrained the usable volume. Future work should focus on producing more concentrated extracts or employing alternative detection methods (e.g., viability staining) to overcome these limitations.

Results: Assessment of Propolis Antimicrobial Activity

We aimed to evaluate the antimicrobial efficacy of propolis extract, prepared using PEG 400 to avoid ethanol-related interference, against Escherichia coli and Saccharomyces cerevisiae. Despite iterative adjustments to the experimental design, consistent inhibition was only observed using a 96-well plate assay, which allowed for quantitative analysis of growth inhibition.

Experimental Approach and Observations

Agar Well Diffusion Method

Initially, the agar well diffusion method was employed, following a published protocol. Propolis extract (assumed phenolic concentration: 10.7 ± 1.2 mg/mL) was applied to wells in dual-layer agar plates at volumes of 100 μL, 150 μL, and 200 μL. After 48 hours of incubation, no clear zones of inhibition were visible, though slight growth reduction was noted near the wells. Repeating the experiment with slightly dried plates and a 20% PEG 400 positive control yielded similar results, suggesting limited diffusion or insufficient antimicrobial activity under these conditions.

Colony Assay Stamp Method

A 3D-printed colony assay stamp was introduced to standardize inoculation and improve diffusion. Plates were stamped with microorganisms, and propolis extract was added to a central well. Post-incubation, colonies closer to the well appeared smaller, indicating a potential localized effect. However, nutrient competition among colonies could not be ruled out as a contributing factor.

96-Well Plate Assay

To enhance repeatability and quantifiability, a 96-well plate assay was implemented. Optical density measurements over time revealed a dose-dependent inhibitory effect: higher propolis concentrations correlated with reduced microbial growth. Controls with 20% PEG 400 confirmed that the solvent alone did not contribute to inhibition. Nevertheless, the effect was modest, likely due to the extract s opacity limiting accurate optical density readings at higher volumes.

![Results](https://static.igem.wiki/teams/5728/illustrations/result3.avif)

*Figure 3. Growth curves of S.cerevisiae in presence of increasing concentrations of Propolis and PEG solution.*

![Results](https://static.igem.wiki/teams/5728/illustrations/result4.avif)

*Figure 4. Growth curves of S.cerevisiae in presence of increasing concentrations of Propolis and PEG solution.*

Discussion

The lack of visible inhibition zones in agar-based methods may stem from insufficient extract diffusion or low active compound concentration. The 96-well plate assay provided clearer evidence of antimicrobial activity, though the opacity of the extract posed a technical challenge. These findings suggest that while propolis exhibits antimicrobial properties, the extract’s potency or the method’s sensitivity may need optimisation for robust detection.

Conclusion

Propolis extract demonstrated antimicrobial activity in liquid culture, but its efficacy was weaker than anticipated. The 96-well plate assay proved more reliable for quantifying growth inhibition, though extract opacity constrained the usable volume. Future work should focus on producing more concentrated extracts or employing alternative detection methods (e.g., viability staining) to overcome these limitations.

Results: phage virulency in proximity to propolis

We evaluated the ability of phages to infect and lyse bacterial host cells in the presence of propolis extract. The propolis extract was prepared using a 20% aqueous PEG 400 solution, to which propolis powder was added at a 1:10 (w/v) ratio. The mixture was heated at 70°C for 15 minutes to facilitate extraction, resulting in an assumed phenolic compound concentration of 10.7 ± 1.2 mg/mL, as previously described by Koo et al.(2015). PEG 400 was selected as the extraction solvent to avoid the bacterial growth inhibition associated with ethanol-based extracts.

To assess the compatibility of phages with propolis, a 96-well plate assay was designed. Host bacterial cultures (Escherichia coli) were first grown in LB media to an optical density (OD600) of 0.4–0.6, corresponding to the exponential growth phase. Once the target OD was achieved, the measurement cycle was paused, and the plate was removed under aseptic conditions. Propolis extract at six different concentrations, along with an active phage culture, was then added to the wells. The plate was reinserted into a TECAN Infinite® M Nano microplate reader, and OD600 measurements were resumed to monitor bacterial lysis over time. The assay included a negative control (propolis extract + media, without bacterial inoculum or phages) and a positive control (host bacteria + propolis, without phages), both following the same propolis concentration gradient as the test wells.

Results and Analysis

Analysis of the data revealed that phages retained their lytic activity across all propolis concentrations tested. Although propolis exhibited a slight inhibitory effect on bacterial growth, this did not significantly impair the phages ability to infect and lyse their host cells. The results indicate that propolis, at the concentrations used, does not interfere with the phages capacity to target and destroy bacteria.

![Results](https://static.igem.wiki/teams/5728/illustrations/result5.avif)

*Figure 5. Data representing OD600 measurements of the 96-well assay for testing phage and propolis compatibility.*

Discussion and Implications

These findings suggest that phages and propolis can coexist without antagonistic effects, supporting the feasibility of their combined use in antimicrobial strategies. The mechanisms underlying phage infection—including receptor recognition, genome injection, and host cell takeover—appear to remain functional in the presence of propolis. This compatibility is particularly valuable, as it opens the door to synergistic applications where propolis could provide broad-spectrum antimicrobial coverage, while phages offer targeted elimination of specific bacterial pathogens.

Conclusion and Future Directions

In conclusion, we have demonstrated that phages can effectively survive, infect, and lyse bacterial cells in the presence of propolis extract. While propolis alone exerts a mild inhibitory effect on bacterial growth, it does not compromise phage activity, making their combined use a viable strategy for enhanced antimicrobial efficacy. Future work could explore the optimization of phage-propolis combinations, particularly against multidrug-resistant bacterial strains, and investigate their stability and activity in more complex environments.

References

[1] D. M. Francis and R. Page, “Strategies to Optimize Protein Expression in *E. coli*,” *Current Protocols in Protein Science*, vol. 61, no. 1, Aug. 2010, doi: https://doi.org/10.1002/0471140864.ps0524s61

[2] F. Song, L.-M. Zhang, C. Yang, and L. Yan, “Genipin-crosslinked casein hydrogels for controlled drug delivery,” *International Journal of Pharmaceutics*, vol. 373, no. 1–2, pp. 41–47, Feb. 2009, doi: https://doi.org/10.1016/j.ijpharm.2009.02.005

[3] L. Kubiliene *et al.*, “Alternative preparation of propolis extracts: comparison of their composition and biological activities,” *BMC Complementary and Alternative Medicine*, vol. 15, no. 1, May 2015, doi: https://doi.org/10.1186/s12906-015-0677-5

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