Project Description

Project Overview

Our project aims to address the urgent challenge of rising global temperatures by improving the resilience of Chlamydomonas reinhardtii. Rather than focusing solely on carbon capture, we will explore how engineered algae can survive and function effectively under heat stress, ensuring that future bioengineering solutions remain viable in a warming world.

To achieve this, we are investigating CPN60C, a mitochondrial chaperonin (heat shock protein 60 family member) that assists in proper protein folding under thermal stress (UniProtKB, I2FKQ9). By overexpressing CPN60C in Chlamydomonas reinhardtii, we aim to enhance the organism’s ability to maintain metabolic stability and photosynthetic activity at elevated temperatures (Malkeyeva, 2025; Gupta, 1995).

Long term, our project seeks to contribute to the development of thermotolerant microalgae that can sustain growth and carbon assimilation in environments increasingly affected by climate change. Beyond the immediate scientific value, this work supports the broader vision of building biological systems that are sustainable and resilient.

The Issues: Global Warming, the Rising Concentration of CO2, and Temperature Stress

Wherever you are in the world -- China, Vanuatu, the United States, France or Germany, just like our team members and friends who support us from across different continents and oceans -- you may have noticed the rising temperatures over the past years and decades. According to the Copernicus Climate Change Service (C3S) report, in 2024, the global average temperature was 1.6 °C higher than during the pre-industrial era.

Such a phenomenon is commonly known as “global climate change”, which is a pressing issue with increasingly devastating consequences. In addition to rising temperatures (“global warming”), it can lead to other severe outcomes, including but not limited to rising sea levels, the loss of biodiversity and ecological balance, the disruption of weather patterns, and threats to the health of humans and other life (Hassan, 2024).

Climate change and global warming are being driven largely by human activities, such as burning fossil fuels and clearing forests, both of which release carbon dioxide and remove natural systems that normally store it. As a result, carbon dioxide, which is the main greenhouse gas contributing to global warming, has increased dramatically in the atmosphere (Hassan, 2024). Microalgae are small, chloroplast‐containing aquatic organisms that capture carbon dioxide through photosynthesis while also producing useful biological compounds (Sun at el., 2025). Studies show that one ton of microalgal biomass can absorb approximately 1.83 tons of carbon dioxide (Ho et al., 2011). Because of this impressive ability to capture and store carbon, microalgae are increasingly seen as a promising tool for mitigating greenhouse gas emissions.

Global warming attribution illustration

Fig. 1. Graph showing global warming attribution - based on NCA4 (2017) Fig 3.3. Modeled forcing responses of forces affecting global temperature (1870 - ). (RCraig09, 2020, via Wikimedia Commons under CC4.0， with no modification)

Nevertheless, temperature stress affects all living organisms, from crops to microscopic algae. For photosynthetic organisms, heat can cause protein misfolding, enzyme inactivation, and oxidative stress, ultimately impairing growth and carbon fixation (Singh, et al., 2024; Vera-Vives, et al., 2024). While enhancing the carbon fixation capability of microalgae is promising, these organisms themselves are vulnerable to rising temperatures. Therefore, it is equally important to ensure that photosynthetic organisms, which are key players in global carbon cycling, can withstand the conditions of a changing climate.

The Inspiration: Why Global Warming, Why C. reinhardtii, and Why CPN60C

The global nature of our team makes climate change an issue that resonates with all of us. Based in Shanghai, China, our school partners with Gaston Day School in North Carolina, United States. We also have teammates from Chongqing, China, a city renowned not only for its hot-pot cuisine but also for its “hot-pot-like” summer climate. Our instructors and advisors likewise come from diverse cities across both China and the United States. When we first brainstormed potential research topics and mentioned “global warming,” the immediate response was unanimous: “Yes, I can feel it!” The same reaction came from our international friends, who echoed the sentiment with identical words: “Yes, I can feel it!”

As we considered how synthetic biology could contribute to addressing this challenge, we were drawn to Chlamydomonas reinhardtii. This unicellular green alga of the Chlorophyta is a well-established model organism in climate and bioenergy research. With its fully sequenced genome, genetic tractability, and high photosynthetic efficiency, it serves as an ideal chassis for studying mechanisms of stress tolerance (Salomé & Merchant, 2019).

Within this system, we focused on CPN60C, a member of the chaperonin 60 (HSP60) family. While other Cpn60 isoforms localize to chloroplasts to facilitate folding of photosynthetic proteins, CPN60C is believed to localize to mitochondria, where it supports respiration and energy metabolism under stress (UniProtKB, I2FKQ9). Previous studies highlight the essential role of mitochondrial chaperonins in protecting cells from thermal denaturation and maintaining cellular homeostasis (Singh et al., 2024; Vera-Vives et al., 2024). We therefore hypothesize that overexpressing CPN60C will enhance the heat resilience of Chlamydomonas by stabilizing key metabolic enzymes and improving recovery following stress exposure.

The Evolution: from LCIB to CPN60C

We initially planned to focus our research on the endogenous gene LCIB (Low-Carbon Dioxide Inducible B) in Chlamydomonas reinhardtii. As Wang and Spalding (2014) pointed out, LCIB plays a significant role in the carbon-concentrating mechanism of Chlamydomonas, being strongly expressed under low-CO₂ conditions. However, through an unexpected slip during our project work, we came across another gene, the mitochondrial chaperonin CPN60C, which caught our attention due to its potential role in protecting algal cells under heat stress. At the same time, during our summer experiments, we noticed that our algal cultures often struggled to survive the hot weather. This raised a practical question: even if a genetically modified algal strain has an enhanced carbon-concentrating mechanism, how can it be applied in regions most affected by rising temperatures if it cannot itself withstand heat stress?

Motivated by this question and our growing curiosity, we decided to shift our research focus from LCIB to CPN60C. Both are endogenous genes in Chlamydomonas, and the principle of overexpression plasmid construction is the same. Compared with LCIB, moreover, it seems that CPN60C has been studied a bit less extensively, which could make our project not only potentially more relevant for real-world applications but also more innovative.

At the same time, we remain strongly interested in LCIB and would welcome the opportunity to study it in the future. We also look forward to seeing more insights into this gene from both the iGEM community and the wider scientific field.

The methodology: overexpression of CPN60C in Chlamydomonas reinhardtii

  1. With guidance and support from external experts (please see the Team Attribution, Experiments and Engineering pages for more details), we designed plasmids carrying the endogenous CPN60C gene of Chlamydomonas reinhardtii under control of the PsaD promoter and terminator as well as the HSP70A-RBCS2 fusion promoter and RBCS2 terminator and got them constructed, ensuring strong and constitutive expression. The plasmid backbones contain selectable markers for antibiotic resistance (paromomycin and hygromycin), allowing efficient screening of transformants.
  2. We employed both wild-type (CC-124) and cell wall-deficient (UVM4) strains as our experimental systems.
  3. The constructs were introduced into algal cells via electroporation and plated on media containing the corresponding antibiotics, incubated, and cultured until reaching the mid-logarithmic phase of growth. This procedure, including both algal culture and transformation, was repeated throughout our lab period.
  4. By culturing the transformed algae on selective plates, we identified the groups of initial potential positive transformants that survived antibiotic selection. PCR and qPCR were subsequently used to confirm the successful overexpression of the CPN60C protein.
  5. For the positive transformants confirmed through the steps above, we subjected them, together with negative control strains, to heat stress tests starting from the same growth stage and under identical conditions. Their growth phenotypes were then observed and measured to assess whether CPN60C overexpression provided protection under heat stress.

References

  • Allakhverdiev, S. I., Kreslavski, V. D., Klimov, V. V., Los, D. A., Carpentier, R., & Mohanty, P. (2008). Heat stress: an overview of molecular responses in photosynthesis. Photosynthesis Research, 98(1-3), 541–550. https://doi.org/10.1007/s11120-008-9331-0
  • Copernicus Climate Change Service. (2025, January 10). Global climate highlights 2024. Retrieved August 14, 2025, from https://climate.copernicus.eu/global-climate-highlights-2024
  • Gupta R. S. (1995). Evolution of the chaperonin families (Hsp60, Hsp10 and Tcp-1) of proteins and the origin of eukaryotic cells. Molecular Microbiology, 15(1), 1–11. https://doi.org/10.1111/j.1365-2958.1995.tb02216.x
  • Hassan, N. E. (2024). Global warming: Causes, impacts and urgent strategies for a sustainable future.
  • Ho, S. H., Chen, C. Y., Lee, D. J., & Chang, J. S. (2011). Perspectives on microalgal CO2-emission mitigation systems—a review. Biotechnology Advances, 29(2), 189-198.
  • Malkeyeva, D., Kiseleva, E. V., & Fedorova, S. A. (2025). Heat shock proteins in protein folding and reactivation. Vavilovskii zhurnal genetiki i selektsii, 29(1), 7–14. https://doi.org/10.18699/vjgb-25-02
  • RCraig09. (2020, February 13). 2017 global warming attribution – based on NCA4 Fig 3.3 – single-panel version [SVG image]. Wikimedia Commons. https://commons.wikimedia.org/wiki/File:2017_Global_warming_attribution_-_based_on_NCA4_Fig_3.3_-_single-panel_version.svg
  • Salomé, P. A., & Merchant, S. S. (2019). A series of fortunate events: introducing Chlamydomonas as a reference organism. The Plant Cell, 31(8), 1682-1707.
  • Singh, M. K., Shin, Y., Han, S., Ha, J., Tiwari, P. K., Kim, S. S., & Kang, I. (2024). Molecular Chaperonin HSP60: Current Understanding and Future Prospects. International Journal of Molecular Sciences, 25(10), 5483. https://doi.org/10.3390/ijms25105483
  • Sun, Z., Bo, C., Cao, S., & Sun, L. (2025). Enhancing CO2 fixation in microalgal systems: Mechanistic insights and bioreactor strategies. Marine Drugs, 23(3), 113.
  • UniProtKB entry I2FKQ9, Chlamydomonas reinhardtii CPN60C. Retrieved from https://www.uniprot.org/uniprotkb/I2FKQ9/entry
  • Vera-Vives, A. M., Novel, P., Zheng, K., Tan, S.-L., Schwarzländer, M., Alboresi, A., & Morosinotto, T. (2024). Mitochondrial respiration is essential for photosynthesis-dependent ATP supply of the plant cytosol. bioRxiv. https://doi.org/10.1101/2024.01.09.574809
  • Wang, Y., & Spalding, M. H. (2014). Acclimation to very low CO₂: contribution of limiting CO₂ inducible proteins, LCIB and LCIA, to inorganic carbon uptake in Chlamydomonas reinhardtii. Plant Physiology, 166(4), 2040–2050.