Contribution

We showed that phages can survive, infect and lyse bacterial cells in the presence of propolis. For propolis extract we used 20% aqueous PEG solution. Propolis powder was added to the PEG solution in proportions of 1:10 w/v, respectively. The solution was heated at 70 °C for 15 minutes, and an approximate phenolic compound content of 10.7 ± 1.2 mg/ml was assumed based on Loreta Kubiliene et al., 2015 [1].

To test if phages keep their ability to infect host cells and lyse them in the presence of antimicrobial active compounds of propolis, a 96-well plate assay was designed. This assay included host bacteria media (LB), host cell culture, propolis extract in differing concentrations, and active phage culture.

To actually monitor how the phages lyse the bacterial host cells, we first grew the bacterial cultures to OD₆₀₀ ~ 0.4–0.6 in media, without propolis or phages. After the respective OD₆₀₀ was reached, the measurement cycle was to be paused, the plate removed, and under aseptic conditions the respective amount of propolis extract and active phage solution added. The plate was to be inserted back into the OD measurement device; in our case, we used TECAN’s Infinite^® M Nano microplate reader and the measurement cycle would be unpaused.

In total, one assay tested 6 different concentrations of propolis extract plus a negative control, which included propolis extract and media without inoculum, and a positive control with host bacteria cells and propolis but no phages, both controls also following the propolis extract concentration gradient.

After analysis of the data in Figure 2, a plot was made that showed that there was no significant yet slight inhibitory effect of propolis on phages' ability to infect and lyse host bacterial cells.

To further test this primer pair, PCR amplification of a 1.8 kb gene fragment was performed, where complementary sequences to both primers appeared only after allowing for 6 mismatches. To assess the specificity of this primer pair, four PCR runs were conducted: three standard PCR runs at increasing annealing temperatures (61°C, 64°C, and 67°C), and one touchdown PCR (tPCR) starting at 71°C, with 1°C decrements down to 61°C. Each standard PCR run had three replicates, while the tPCR had five replicates. The composition of the PCR reagent mixes was identical and prepared simultaneously, using Phusion polymerase.

After the PCR program, all samples were loaded onto an agarose gel and subjected to electrophoresis. Following electrophoresis, the gel was visualized and conclusions were drawn.

The results indicated that, while some non-specific amplification occurred, it was relatively minor compared to the amplification of the target amplicon. Notably, non-target amplification was completely eliminated at an annealing temperature of 67°C. Observations also suggested that tPCR did not improve the specificity of the primer pair.

From these findings, we concluded that our universal primer pair functions effectively at higher temperatures and that increasing the annealing temperature enhances their specificity.

As we performed our first colony PCRs, we noticed that we were not able to see amplicons because, at the same length, in the electrophoresis gel, a bright “cloud of doom” was present. At first we believed that it was some sort of cell debris or something else in the PCR product left over from cell lysis. We figured that if most of the interfering material could be centrifuged off, the bands of our target amplicons could be seen. One big issue with that plan was that we didn’t have a way to centrifuge 0.2 ml tubes at high speeds. To solve this issue, we decided to design and 3D print adapters for the small PCR tubes. It was designed so that the adapter would fit into a standard 2 ml or 1.5 ml centrifuge tube carrier.

Microsoft’s “3D Builder” was used to design the adapters by first modelling a 2 ml tube. Then a 0.2ml tube was also designed. The 2 models were aligned on top of each other so that the 0.2 ml tube model would sit in the center of the 2ml tube model. After alignment, the 0.2ml tube model was subtracted from the 2ml tube model, creating a place where the 0.2ml tube would sit in.

This model was saved as an .stl file, then Mango 3D’s “Lychee Slicer” was used to slice the file and generate a .pmx2 file that our 3D printer would recognise. We used the Anycubic Photon Mono X2 resin 3D printer with Anycubic’s Tough Resin 2.0 3D printing photopolymer material.

After printing, parts were washed in an ultrasonic bath with ethanol to remove any residual resin. Then parts were fully cured for 1 hour under a UV (365-405 nm) light source.

For the first prototype, the 0.2 ml tube did not fit the adapter and sat halfway above the intended place. We concluded that we had forgotten to add allowances since, during the design process, the exact measurements of the 0.2 ml tube were used.

This was a simple fix, though – we went back to the modelling software and increased the size of the 0.2 ml tube model by 1 mm on all axes (X, Y and Z axes). Then, models were aligned again, and the 0.2 ml tube model was subtracted. Further on, the same steps were used as last time for model preparation and printing and downstream preparation. This time, 0.2ml tubes fit perfectly.

To test these adapters, we spun down a dense E. coli cell culture, and the process seemed successful – supernatant was clear, and a small pellet was observed.

To test the capabilities of the 3D-printed adapter, we loaded some 0.2 ml tubes with 200 µl of H₂O, inserted them into the adapters, and inserted the adapters into a centrifuge carrier. The first high-speed run was at 10000 rpm for 5 minutes; the adapters appeared undamaged after. Time was increased to 10 minutes, and again, adapters seemed undamaged after the centrifugation. Now, speed was increased to 15000 rpm, as fast as our centrifuge would go, and time was set for 10 minutes. After centrifugation, adapters showed no visible damage, and 0.2 ml tubes were still in place.

The 3D-printed adapters were deemed to be safe for use in the laboratory, and more were printed for future use.

After unsuccessful protocol with antimicrobial tests of propolis extract with the agar well diffusion method with dual-layer agar inoculation, we figured that another approach could be taken.

Our plan was to design a press that would transfer colonies in a certain pattern from a fully colonised plate, or liquid media, onto another, new agar plate.

A simple design was made, suitable for a Petri plate with a diameter of 86 mm or above. A round bottom plate with arranged blunt spikes was designed in Microsoft’s “3D Builder” software. Additionally, a simple handle was also designed, so after printing the models, it could be glued to the back of the presser for easier handling. The choice was made to glue the handle after printing both models separately because adding a handle pre-print would cause issues with positioning the models in a way that allows successful printing.

The model was saved as an .stl file, and then Mango 3D’s “Lychee Slicer” was used to slice the file and generate a .pmx2 file that our 3D printer would recognise. We used the Anycubic Photon Mono X2 resin 3D printer with Anycubic’s Tough Resin 2.0 3D printing photopolymer material.

After printing, parts were washed in an ultrasonic bath with ethanol to remove any residual resin. Then parts were fully cured for 1 hour under a UV (365-405 nm) light source. The handles were glued to the back of the plates using cyanoacrylate adhesive.

These devices were tested by immersing the spiky side in a liquid culture that had been poured in a petri dish lid and pressing on a new agar plate and incubating overnight at the respective temperature.

Additionally, another method was tested – by growing a lawn on an agar plate with a dual-layer agar technique, then gently lowering the presser onto the colonised agar surface and re-pressing on a new agar plate, also incubated overnight after inoculation.

All 3D files, both for 0.2ml adapters and Colony Assay stamps are available here.

This year, we developed The Constellation of Care — a practical well-being and teamwork guide designed to help both our team and future iGEM teams build sustainable, healthy working environments. Drawing from our own experiences, challenges, and lessons throughout the season, we created a set of structured exercises called The Five Cosmic Pillars: Gravity, Orbit, Fuel, Stars, and Constellation. Each pillar focuses on a key aspect of effective collaboration — responsibility, communication, well-being, trust, and reflection — forming the foundation of a balanced and resilient team.

Our guide includes activities such as responsibility mapping to prevent workload imbalance, weekly stand-ups to keep communication open, the WHO-5 burnout self-test and mindfulness exercises to support mental health, 1:1 trust check-ins to strengthen connections, and after-action reviews for continuous learning. Together, these practices create a framework that teams can adapt to their own rhythm, helping members recognise early signs of burnout, maintain motivation, and manage projects sustainably.