Our part collection includes a wide variety of synthetic genes designed specifically for the metabolic pathways of Chlamydomonas reinhardtii, and optimized so the sequence is compatible with the microalgae and contains minimal quantities of GC.:

Nitrates Sensor

This is a new part that indicates the changes in nitrate concentration of the medium. It is characterized by:
- Lack of antibiotic resistance gene linked to the reporter.
-Includes three cassettes (pCM1s), each with a constitutive nitrate-dependant promoter: mVenus, mCherry, and HygroR.
All three cassettes combined create the Level M of the sensor.
In the wet lab, we have thus far been able to demonstrate that our custom nitrate sensor successfully transformers and assembles both in Level 1 and Level M in E. coli.
Afterwards, we were also able to demonstrate the sensor transforms in Chlamydomonas reinhardtii.

Pathway genes

Our part collection includes two basic sets of genes.
- Set 1, Nitrate assimilation: Nitrate Reductase (NR), Nitrites Reductase (NiR), Glutamate Synthetase (GS), Cp-GS
- Set 2, Cytokinin production: IPT, LOG
Vectors containing the genes above are referred to as “pathway genes”.

Level 1

Our Level 1 genes are designed to overexpress the genes they contain.
All of our genes, except Cp-GS, include a special-designed Level 1 that includes a PPSAD promoter, a coding sequence, an F2A peptidase, a NanoLuc reporter and a TPSAD terminator.
Cp-GS’ Level 1 is characterised by the presence of a chloroplast transit peptide.

Level M

Structure of four cassettes that include: resistance, gene, mVenus and mCherry. The basic composition is very similar to our Level 1.
The Level Ms may contain individual genes, paired genes or a combination of genes. The amount of genes will change the position of the cassettes.
Each of the Level M types has a different function.

Mo-Clo collection

After designing the cloning plans, we adapted the sequences for MoClo. The special features in our Mo-Clo collection part are:
- Added unique overhangs to assemble genes to the backbone vector
- Eliminated recognition sites for BbsI and Bsal
After using our personal software to optimize the codon sequence of our genes so they are synthesizable (which has proven to be a great challenge before due to their high GC content). We also confirmed aminoacid sequences of transformed genes and our engineered genes were the same.
Our Mo-Clo collection, therefore, includes our customized nitrate sensor and Chlamydomonas reinhardtii genes NR, NiR, GS, Cp-GS, LOG, and IPT which are compatible with Mo-Clo technology and can be commercially synthesized.

All these pathway genes have been able to assemble into Level 1s successfully.

We developed a software that could reduce the high local GC content and the high repeated sequences. This is a common problem many scientists face when designing genes in Chlamydomonas so they can be ordered and used in specific experiments. With the aid of our code, we could reduce the number of repeated sequences and achieve an acceptable content of GC without changing the gene information and regulation.

It proved to be really useful for us when we had to design our constructs. Due to the characteristics of the genes we wanted to work with, we faced this problem several times and, since changing the nucleotides by hand was an extremely arduous task, we developed this method to achieve a more efficient construct design. Our constructs ended up being correct and valid so they could be synthesized.

As our experiment is highly dependent on the relation between Chlamydomonas reinhardtii and nitrate-rich mediums, we had to design a set of experiments to test the compatibility of algae growth in different nitrate concentrations.

We had a fixed set of concentrations we worked with: 1 mM, 15mM, 45mM, 90mM, 105mM, 280mM and 385mM. Mediums of 90 and above are not commonly found in fresh waters, as they are very high concentrations of nitrate, but we still wanted to test them since our photobioreactor with our genetically engineered Chlamydomonas reinhardtii is intended for highly nitrate-polluted areas.

Our main intention was to test the highest concentration of nitrates in the medium to determine both when they stopped being functional and what their optimal working concentrations were.

We have determined that 1mM and 15mM nitrate concentrations are the best performing, whilst 105mM concentrations generate clumps and facilitate algae precipitation.
Additionally, we very quickly realised that 280mM and 385mM nitrate concentrations are mortal for Chlamydomonas algaes.
Still, the ability of the microalgae to tolerate and function in 105mM concentration is significant, and means the algae has high compatibility for nitrate rich mediums.

Therefore, for future experiments, researchers could benefit from the properties described and tested and start also implementing Chlamydomonas as a useful organism to treat high-nitrate concentrated waters.

1.Some individual components boost algae growth

First, we determined how we can increase Chlamydomonas reinhardtii growth rate when cultured in TAP medium supplemented with different components in comparison with their growth in just TAP.
Choosing the components under the criteria that they could be found in wastewater, we observed how some of them work as growth inhibitors and others stimulate their growth rate above the control.
- Inhibitor: Na2EDTA·2H2O
- Stimulators:
ߦUVM4 strain: CaCO3, NaCl, KH2PO4, MnCl2·4H2O, CoCl2·6H2O, FeCl3·6H2O and CaCl2·2H2O (from highest growth boost to lowest growth boost above the control sample).
ߦLM8523 strain: CaCO3, MnCl2, NaCl, CoCl2·6H2O, KH2PO4, FeCl3·6H2O, MgSO4·7H2O, ZnCl2 (from highest growth boost to lowest growth boost above the control sample).
Moreover, medium supplemented with FeCl3·6H2O, MgSO4·7H2O, ZnCl2 had not yet reached the growth peak even at 168h.

Therefore, we have found cheap components that could be added to Chlamydomonas reinhardtii classical culture medium and can boost their growth. They are easy to obtain, hence they are a promising option for other research experiments in which they want to culture C. reinhardtii and get them fully grown in a short period of time.

This a new finding, since it has always been thought that TAP medium is the best option to culture Chlamydomonas, but we’ve proven that the mediums can be improved so the growth rates are accelerated.

Those supplemented mediums could be used by other iGEM teams that plan to work with C. reinhardtii, research groups in microalgae, and implement our bioreactor and make nitrate absorption and cytokinin production even more efficient.

We focused on kinetin, a type of cytokinin, to address salinity stress in plants, especially for the purpose of enabling the use of moderately salty water for crop irrigation in areas with high water stress. This idea is both innovative and highly relevant and it can contribute to:
- Salinity mitigation: we contributed to the understanding of how cytokinins can mitigate the adverse effects of salinity. This small investigation could lead to the development of strategies to make agriculture more sustainable in regions with salinity issues. We can also enhance crop resilience.
- Local relevance: these findings have direct relevance to the local community, especially those that face salinity problems in their waters
- Environmental sustainability: the use of moderately salty water for irrigation can be a more sustainable approach, especially in regions where freshwater is scarce or at risk of contamination.
- Research and knowledge building: we contribute to the body of knowledge on plant responses to salinity stress, particularly in the context of cytokinin treatment. This study can serve as a foundation for future studies and innovations in agriculture.