Results

Abstract

Our project aimed to construct a plasmid carrying the endogenous CPN60C gene and transform it into Chlamydomonas reinhardtii, thereby achieving overexpression of this endogenous gene. The objective was to improve the survival and photosynthetic efficiency of Chlamydomonas reinhardtii under heat stress, providing a potential green strategy to mitigate global warming.

The experimental results demonstrated the following:

• Using paromomycin and hygromycin as selective antibiotics, 10 μg/mL was identified as the optimal resistance screening concentration under our experimental conditions, particularly in electroporation of cell wall–deficient mutant strains.
• Compared with the cell-walled, wild-type CC-124 strain, the cell wall–deficient mutant UVM-4 exhibited markedly higher transformation efficiency. However, owing to its cell wall defect and the additional metabolic burden associated with CPN60C overexpression, UVM-4 was prone to chlorosis (cell death), rendering it unsuitable for large-scale industrial or practical applications.
• PCR and qPCR analyses shows that both plasmids successfully transformed CC-124 into positive transformants, each exhibiting different levels of target gene upregulation.
• Heat stress experiments revealed that, compared with wild-type CC-124 and transformants with only marginal increases in expression, transformants with substantially elevated CPN60C expression displayed significantly enhanced survival under heat stress.

In summary, our findings provide preliminary evidence that overexpression of the endogenous CPN60C gene enables Chlamydomonas reinhardtii CC-124 to remain viable at elevated temperatures (40 °C). This suggests that Chlamydomonas reinhardtii could sustain robust growth and efficient photosynthesis in hot environments, thereby strengthening its potential for large-scale industrial use and urgent real-world applications, and enhancing its value and feasibility as a biological solution to global warming.

1. Antibiotic Sensitivity Assay

Our experimental results showed that hygromycin and paromomycin completely inhibited the growth of untransformed negative control algae strains (UVM-4 and CC-124) at all three tested concentrations: 5 μg/mL, 10 μg/mL, and 20 μg/mL. These findings confirm that, under our experimental conditions (see the Experiments page for details), these concentrations are sufficient for resistance screening and can effectively distinguish potential positive transformants from negative controls.

2. Optimal Resistance Screening Concentration

Under our experimental circumstances, although all three antibiotic concentrations effectively suppressed the growth of negative control algae, the transformation experiments demonstrated a marked decline in the growth density of potential positive transformants with increasing concentration:

At 5 μg/mL, the plate was densely covered with algal colonies (Figure 1A);

At 10 μg/mL, colony growth was markedly reduced (Figure 1B);

At 20 μg/mL, only 2–3 colonies were observed on the plate (Figure 1C).

In studies of Chlamydomonas reinhardtii, paromomycin concentrations of 10–40 μg/mL are generally considered suitable for resistance screening (Chlamydomonas Resource Center, n.d.). For hygromycin, some studies have reported that 15 μg/mL is sufficient to completely inhibit algal cell growth (Qin et al., 2021), while 10 μg/mL has been used as a working concentration for the transformation of cell wall–deficient strains (Chen et al., 2015).

In our experiments, although 5 μg/mL was sufficient to inhibit the growth of all negative control strains, we considered the potential risk of false positives at such a low concentration. Conversely, 20 μg/mL greatly restricted the growth of transformed colonies. Since our study involved both the wild-type CC-124 strain (with intact cell walls) and the cell wall–deficient UVM-4 strain, we also needed to account for UVM-4’s reduced tolerance to higher antibiotic concentrations while keeping the experimental conditions as consistent as possible.

Therefore, to balance rigorous selection with high transformation efficiency, we ultimately chose 10 μg/mL as the optimal concentration for resistance screening.

3. Effect of Algal Strain Genetic Background on Transformation Efficiency

Under optimized selection conditions with 10 μg/mL hygromycin, the transformation efficiency of the cell wall–deficient strain UVM-4 (>300 colonies per plate) was markedly higher than that of the wild-type strain CC-124 (>100 colonies per plate), which retains an intact cell wall. These results are presented in Figure 2.

We attribute this result to the physiological characteristics of UVM-4, which lacks a functional cell wall. This defect facilitates the uptake of endogenous DNA and leads to higher transformation efficiency. Our findings are consistent with previous studies showing that UVM-4 exhibits a higher transformation rate than wild-type Chlamydomonas reinhardtii strains with intact cell walls (Geisler et al., 2021; Neupert, Karcher, & Bock, 2009), further explaining why UVM-4, as a laboratory mutant, has received considerable attention in the Chlamydomonas reinhardtii research community.

4. Effect of Strain Genetic Background on Viability

4.1 Cultivation of Negative Control Strain

When expanded under identical experimental conditions, the wild-type strain CC-124 exhibited excellent viability and successfully preserved cultures. In contrast, the cell wall–deficient strain UVM-4 did not survive, leading to failure to preserve the culture.

Preliminary analysis suggests that these results are closely related to our initial continuous-light (24-hour) cultivation strategy. While this approach accelerates algal metabolism and allows the strains to enter the logarithmic growth phase within 3–5 days, it may also cause them to enter the decline phase prematurely. Because of its defective cell wall, the UVM-4 strain is more sensitive to environmental stress and physiologically fragile. Although this feature enables higher efficiency in endogenous gene transformation, it also makes UVM-4 easier to decline and die under continuous-light stress, resulting in the failure to preserved culture.

Improvements: To prevent recurrence of this issue, the following optimization measures will be applied in subsequent cultivations:

First, single algal colonies will be reisolated from the original strain for expansion, providing algal colonies that are physiologically healthy at the beginning of the experiments.

Second, the lighting regime will be shifted from continuous light to a 16-hour light/8-hour dark cycle to better mimic natural rhythms, reduce metabolic stress, and support long-term growth vigor and stability.

4.2 Cultivation of Transformed Strains

As shown in Figure 4, the UVM-4 strain transformed with the CPN60C gene displayed a marked growth disadvantage compared with the untransformed negative control UVM-4 strain. In contrast, both the wild-type CC-124 strain and its transformed counterparts exhibited robust growth. During continued cultivation, the transformed UVM-4 strains gradually turned yellow (chlorosis) and died before reaching the logarithmic growth phase.

We speculate that this consequence is attributable to inherent physiological defects of the UVM-4 strain. Although its cell wall deficiency facilitates the introduction of endogenous DNA and thereby increases transformation efficiency, this mutation is also linked to heightened sensitivity to culture conditions and more demanding nutritional requirements. As Schroda & Remacle (2022) noted in a recent review, UVM-4 is less robust and exhibits relatively poor stability.

In addition, the introduction and expression of the endogenous CPN60C gene may further disrupt cellular homeostasis. CPN60C is a mitochondrial chaperone protein that assists in protein folding and protects cells under stress by binding ATP, helping Chlamydomonas reinhardtii survive and photosynthesize under high-temperature stress (Malkeyeva, 2025; Gupta, 1995). However, under non-stress conditions, elevated CPN60C expression may place extra burdens on cellular energy metabolism, potentially leading to growth arrest and cell death in this physiologically fragile strain subjected to metabolic stress.

Based on these findings, and to ensure the feasibility of subsequent molecular verification and functional studies, we selected the wild-type CC-124 strain, with robust growth and greater tolerance to genetic transformation, for PCR verification and related experiments.

5. PCR Verification of Transformed CC-124 Algae

As shown in Figure 5, lanes 1 and 2, as well as lanes 5 and 6, display clear bands with a molecular size of approximately 500 bp. Lanes 1–3 correspond to CC-124 transformed with plasmid A, and lanes 4–6 correspond to CC-124 transformed with plasmid B.

The observed band sizes are consistent with the expected size of the designed specific amplification product, preliminarily confirming that the target fragment was successfully integrated into the algal genome or maintained within the transformant.

Based on these gel electrophoresis results, positive samples with clear and highly specific bands will be selected for real-time quantitative PCR analysis.

6. qPCR Verification of Transformed CC-124 Algae

Real-time quantitative PCR analysis further confirmed successful expression of the CPN60C gene in the transformed strains. As shown in Figure 6, both independent transformants exhibited significant upregulation of CPN60C expression compared with the wild-type CC-124 strain (expression level set as baseline 1). Specifically, the strain transformed with plasmid A (CPN60C-1) showed a relative expression level of 1.44 ± 0.14, approximately 1.44-fold relative to that of the wild type. The strain transformed with plasmid B (CPN60C-2) exhibited an even higher expression level of 2.01 ± 0.25, nearly double that of the wild type.

The qPCR results were consistent with expectations, confirming that the endogenous CPN60C gene was successfully integrated and expressed at the transcriptional level. The observed differences in expression levels between the two transformed algal lines may be attributed to distinct insertion sites of the endogenous gene within the Chlamydomonas reinhardtii genome, or to inherent differences in copy number or transcriptional regulatory element strength between plasmids A and B.

CPN60C is a mitochondrial chaperone protein belonging to the HSP60 family, playing a central role in mitochondrial protein folding, assembly, and stress responses (The Phytozome Consortium, n.d.; UniProt Consortium, n.d.). The upregulation of CPN60C expression observed here suggests that the transformed algal strains may be experiencing specific metabolic or stress states. Moderate overexpression (e.g., 1.44-fold in CPN60C-1) may help maintain mitochondrial protein homeostasis, whereas higher overexpression (e.g., 2-fold in CPN60C-2) could exert stronger effects on mitochondrial function, providing valuable clues for subsequent studies on their physiological roles.

Next, we will investigate the performance of these two algal lines, which exhibit different expression levels, in relation to key physiological indicators of heat stress tolerance.

7. Heat Stress Experiment Results

After subjecting the strains to heat stress by gradually increasing the temperature to 40 °C and then returning them to standard culture conditions, the different algal strains exhibited marked phenotypic differences (Figure 7).

The wild-type CC-124 strain showed significant chlorosis by the second day after heat stress, followed by extensive cell death. This indicates that its photosynthetic system and cellular structure sustained irreversible and severe damage under heat stress.

The low-expression line CPN60C-1, transformed with plasmid A, successfully expressed the endogenous gene; however, its phenotype was similar to that of the wild type, displaying chlorosis and rapid cell death within a short time frame. This suggests that a 1.44-fold relative increase in expression is insufficient to confer effective heat protection.

In contrast, the high-expression line CPN60C-2, transformed with plasmid B, exhibited markedly enhanced stress tolerance. Following the same heat stress, this line largely maintained its green coloration and substantial vitality throughout the recovery culture period, persisting until the end of the observation window (more than two days).

8. Experimental Shortcomings and Next Steps

Over the course of this project, our team encountered numerous challenges but responded swiftly and adapted flexibly, turning obstacles into new opportunities for scientific exploration. For instance, our initial plan was to investigate the LCIB gene, which plays a direct role in the carbon-concentrating mechanism of Chlamydomonas reinhardtii. However, due to coding changes introduced in the gene database update, we selected the CPN60C gene by accident, which functions in the mitochondria. After promptly conducting background research, we determined that this gene offered greater innovation and practical research value. We quickly adjusted our experimental direction, shifting our focus to CPN60C while retaining the same objectives and basic methodologies, and incorporating heat stress experiments.

However, as a mitochondrial chaperone protein that supports algal cell growth, CPN60C may impose an additional metabolic burden under non-stress conditions. Our initial failure to correctly identify the target gene limited our ability to effectively address premature chlorosis of transformants (particularly UVM-4 transformants), which in turn created algal preservation difficulties and insufficient sample volumes for large-scale and repeated PCR/qPCR validation.

In addition, midway through the project, we faced the relocation of our laboratory due to a school-level decision, leaving our PCR system and other key instruments largely unavailable during later stages. To address these constraints, we sought alternative strategies whenever possible, such as monitoring pH changes instead of OD, and collaborating with nearby institutions to complete critical follow-up steps.

Of course, beyond external factors, we also faced challenges stemming from our own technical limitations, gaps in experience, and occasional errors in background research or experimental operation. For example, the electroporation wasn’t always successful; transformants were sometimes contaminated by bacteria and became unusable; and PCR attempts in our own lab at the early stage involved repeated setbacks. Overall, one of the main subjective challenges (distinct from external factors) was achieving more efficient Chlamydomonas culture and transformation while maintaining the cleanliness and viability of potential positive transformants. We hope that future iGEM teams working with Chlamydomonas will benefit from our experiences and lessons learned.

Looking ahead, we plan to strengthen the scientific rigor of our work through the following steps:

• Further preservation and expansion of the negative control strain and the currently healthy CC-124 positive transformants, as well as generating additional positive transformants to enable replicate experiments with larger sample sizes;
• Additional PCR amplification and qPCR data collection;
• Western blot analysis to detect CPN60C protein expression and further confirm overexpression of the target gene in positive transformants;
• Optimization of heat stress assessment through OD measurements;
• Design and implementation of pH measurements consistent with the principles of our earlier experiments.
• Vary environmental conditions to observe potential phenotypic differences, according to Dr. Yuyong Hou's suggestions in our interview.

References

• Chen, L., Li, L., Gao, L., Zhou, J., Li, T., & Peng, H. (2015). Construction and application of the RNAi vector for BBS1 gene in Chlamydomonas reinhardtii. Biotechnology Bulletin, 31(7), 109–114.
• Chlamydomonas Resource Center. (n.d.). pAphVIII (pPH075). Retrieved October 2, 2025, from https://www.chlamycollection.org/product/paphviii-pph075
• 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
• Geisler, K., Scaife, M. A., Mordaka, P. M., Holzer, A., Tomsett, E. V., Mehrshahi, P., ... & Smith, A. G. (2021). Exploring the impact of terminators on transgene expression in Chlamydomonas reinhardtii with a synthetic biology approach. Life, 11(9), 964.
• 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
• Neupert, J., Karcher, D., & Bock, R. (2009). Generation of Chlamydomonas strains that efficiently express nuclear transgenes. The Plant Journal, 57(6), 1140-1150.
• Qin, X. Y., Li, F., Wang, S. J., & Liu, Z. Y. (2021). Optimization of Agrobacterium-mediated transformation of Chlamydomonas reinhardtii. Acta Microbiologica Sinica, 61(1), 92–103.
• The Phytozome Consortium. (n.d.). Cre06.g309100 in Chlamydomonas reinhardtii CC-4532 v6.1. Retrieved October 2, 2025, from https://phytozome-next.jgi.doe.gov/report/gene/CreinhardtiiCC_4532_v6_1/Cre06.g309100_4532
• UniProt Consortium. (n.d.). I2FKQ9 (entry). In UniProt. Retrieved October 2, 2025, from https://www.uniprot.org/uniprotkb/I2FKQ9/entry