Project Overview

Our 2025 wet lab campaign focused on two connected objectives: enabling reliable Chlamydomonas reinhardtii cultivation for future iGEM teams and demonstrating that our engineered strain can act as an efficient rare earth element (REE) biofilter. Throughout the season we iterated on growth conditions, nutrient media, and transformation strategies to remove major experimental bottlenecks.

During our experiments we aimed to :

Refining Cultivation Protocols

For the first goal we were building upon the results of the Chlamy guide (iGEM Kaiserslautern & iGEM Paris Sorbonne 2019). We tested and curated their best techniques to further test their effectiveness. For example most of our experiments were done on their TAP medium, and we measured the amount of cells in liquid media following their procedure. We then further analysed some data to test the growth of the algae depending on additional factors such as temperature and air humidity.

Temperature Screening

As can be seen from our data the optimal growth temperature seems to be at 25°C, but 30°C was nearly equally as good until later on during the experiment. We suspect that the additional stress of higher temperatures adds up and only shows its deleterious effects later on during the experiment. The cell density was measured by taking aliquots of the liquid culture, measuring the optical density on comparing this to the tabulated values we had from counting the cells with a hemocytometer for given optical densities.

The results of this plot can be summarised by directly comparing the growth rates :

Humidity and pH Insights

However our analysis of the growth across different humidity levels did not yield any statistically relevant results. This is not very surprising as our cells were in liquid medium (so not in urgent need of additional H2O). The only beneficial point is that at higher humidity, the liquid medium is less volatile and will stay full for longer. This can be interesting for CO2 media that can feed algae on longer time spans, but it of limited use on TAP medium as it runs out of nutrients relatively quickly after 2 weeks. An additional factor is that higher humidity facilitates fungal growth, which is a problem when growing algae, as funghi also strive very well on TAP-medium. (Applied Microbiology article on TAP-medium contamination)

These results and those of papers investigating the optimal pH for C.reinhardtii growth. (C. reinhardtii pH optimisation study) Motivated our choice of building a bioreactor, to monitor the temperature and pH on large scale C.reinhardtii cultures. To make these advances available to future iGEM teams we also made the construction of it as easy to follow and cheap as possible (see Hardware part).

Medium Development

At the recommendation of Dr. Abiusi, we prepared a slightly modified version of the 6xP Medium (the CO₂ medium). Since CO₂ serves as a carbon source, this medium offers advantages in terms of contamination control and tends to be slightly acidic due to dissolved CO₂.
In the absence of a direct CO₂ supply, we improvised by using balloons, tubing, and a clamp. This setup is not ideal, as the balloon does not release CO₂ evenly or continuously throughout the day. Nevertheless, the algae grew well, indicating that the medium likely also performs effectively in a bioreactor environment.

Knowing that rare earth element (REE) phosphates have low solubility and tend to precipitate, we conducted qualitative experiments to test this behavior. We enriched both our standard 6xP medium and a phosphate-free variant with 100 ppm of REE salts. As expected, precipitation occurred in the phosphate-containing medium, while no visible change was observed in the phosphate-free version. This experiment confirmed that using a phosphate-free medium is a justified and effective choice.

Autolysin Extraction

We also tried a novel approach to C.reinhardtii transformation. As the chlamy guide only investigates glass beads mediated cell wall disruption, that has a high death rate. Autolysin can be used as a natural way to digest the cell wall using an enzyme that C.reinhardtii produces in mating phase. This enzyme is hardly commercially available, so the production and isolation had to be done from our own C.reinhardtii strains. This proved to be unsuccessful as our aliquots of extracted autolysin were not more efficient than the control to digest the cell wall.

We suspect this to be due to our strains. CC5415 and CC124 (mating type + and -) are strains that were selected for their transformation efficiency, not their mating efficiency. Usually to isolate autolysin strains with very high mating efficiency are preferred. We did observe mating (see the cell clumps on the image below) but not as densely as reported in the protocol.

Cassette assembly and transformations

Designing our cassette was quite a struggle, as C.reinhardtii can be quite picky about its genetic sequences it wants to express (see Engineering part) but our plasmid fragments successfully went through assembly simulations.

Although in the Lab we saw something else, as all our sequencing showed defects on our plasmid. The first Golden Gate assemblies failed, and after additional fine tuning of our sequence failed to yield results. We decided to try to make the problematic fragments bind first in a Gibbson assembly. For this we designed primers with overhangs to the backbone on our homologous arms for C.reinhardtii transformation. But even this somehow led to very dirty gel electrophoresis that forced us to proceed with gel extraction.

The following golden gate assembly did yield some transformed bacteria, but sequencing showed that it was always only fragments of our cassette that were integrated and even after multiple attempts no clean product could be isolated, and no advisor knew how to solve the problem.

In an attempt to do more trouble shooting, we tried to send to sequencing our starting material and some intermediates.

The sequencing shows that the PCR gel extraction to introduce the overhangs for Gibbson assembly yielded quite unspecific DNA material which probably interfered with our different experimental steps.

The starting material however seemed to be fine.

The origin of the problem is unsure but it the most probable case would be the large amount of introns that are necessary for an efficient protein production in our transformed algae, that display a very high GC content and therefore also have large repercussions on the efficiency of most synthetic steps. For example the melting temperature was so high that we had to go up to 72°C for our PCR with high fidelity Q5, which is at the very edge of its working range.

In general these high GC content regions that are very common in the C.reinhardtii genome are known to cause problems. Special additives to buffer to increase PCR efficiency in their presence exist, but we did not manage to get one as they are not widely available.

In our last engineering cycle we decided to sacrifice the introns and some additional genes (see cycle 3 in Engineering), these simplification allowed us to get a small enough plasmid for it to be ordered already assembled. The transformation of it happened without a problem on the 3rd of october and the sequencing results confirming these results arrived on the 6th of october. We therefore had no time anymore to purify our plasmid for C.reinhardtii transformation. But this would have been the very next step.

Bioreactor Hardware Integration

In parallel with our biological experiments, we developed a modular, low-cost bioreactor designed to enable scalable and controlled cultivation of Chlamydomonas reinhardtii. The system was engineered to precisely monitor and regulate both temperature and pH.

The reactor is primarily composed of off-the-shelf components and 3D-printed parts, and features the following elements:

• Transparent acrylic cultivation chamber (>10 L) for optimal light transmission and easy cleaning
• LED illumination modules tuned to the spectral range optimal for algal growth
• Integrated sensors (pH and temperature) connected to a Raspberry Pi controller
• Custom software for data logging and automated regulation
• Modular, tripod-mounted design for easy scalability and maintenance

According to the Grand Jamboree safety regulations, no wet testing with non-sterilizable equipment was permitted. Therefore, only dry and functional tests were conducted so far, confirming stable electronic control and reliable sensor feedback.

The bioreactor is now ready for biological integration and future optimization with fully sterilizable components.

This hardware system directly supports both main objectives of our project:

• It provides an open, modular platform for cultivating C. reinhardtii for future iGEM teams.
• It lays the foundation for quantitative bioaccumulation and bioremediation studies using our genetically engineered algae.