Experiments

1. Activation, Expansion, and Cultivation of Chlamydomonas reinhardtii

Our project employed two Chlamydomonas reinhardtii strains commonly used in transgenic studies: the wild-type CC-124, which retains an intact cell wall, and the laboratory mutant UVM-4, which lacks a cell wall and photosynthetic capacity. The primary objective was to overexpress the endogenous mitochondrial chaperone protein CPN60C in C. reinhardtii, while also documenting and comparing differences in cultivation, transformation efficiency, survival, and related aspects. UVM-4 offers notable advantages in transformation efficiency compared to wild-type strains (Neupert, Karcher, & Bock, 2009), so that may serve as a valuable reference and supplement to the wild-type CC-124 in our study.

1.1 Activation and Cultivation of UVM-4

Strain and Culture Medium:
The cell-wall-deficient C. reinhardtii strain UVM-4 was kindly provided by Dr. Lei Zhao and Dr. Yuyong Hou’s team at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. Solid cultures were prepared using TAP medium supplemented with 1.5% (w/v) agar, while liquid cultures were grown in standard Tris–Acetate–Phosphate (TAP) medium (Chlamydomonas Resource Center, n.d.).

Culture Method:
Building on the classical approach described in previous studies (Barolo et al., 2022), we introduced minor optimizations. Briefly, a single colony was scraped from a fresh UVM-4 solid plate with a sterile inoculating loop and transferred into a 100 mL Erlenmeyer flask containing 30 mL of sterile TAP medium. Cultures were incubated at 25 °C under continuous illumination (50–100 μmol photons m⁻² s⁻¹) without shaking.

Growth Monitoring:
Following the method of Chioccioli, Hankamer, & Ross (2014), algal growth was assessed daily by measuring the optical density at 750 nm (OD₇₅₀) with a spectrophotometer. Cells were harvested for subsequent experiments once OD₇₅₀ reached 0.4–0.8, corresponding approximately to the mid-logarithmic growth phase.

1.2 Propagation of CC-124 Chlamydomonas reinhardtii

Strain and Culture Medium:
The wild-type Chlamydomonas reinhardtii strain CC-124 used in this study was purchased from the China General Microbiological Culture Collection. Propagation was carried out in standard Tris–Acetate–Phosphate (TAP) liquid medium.

Propagation Method:
Standard laboratory procedures described in previous studies were followed with minor modifications (Kumar et al., 2004; Haire et al., 2018). Under aseptic conditions, the purchased CC-124 liquid seed culture was inoculated into a sterile conical flask containing fresh TAP medium at a volume ratio of 1:5. For example, 5 mL of seed culture was transferred into 20 mL of fresh medium for a total working volume of 25 mL.

Culture Conditions and Growth Monitoring:
Culture conditions and growth monitoring were identical to those used for UVM-4.

1.3 Plate Streaking Method for Chlamydomonas reinhardtii

Following the standard procedure described by Harris (2009), under aseptic conditions, a sterile inoculating loop was used to pick algal cells in the logarithmic growth phase. The cells were streaked across the surface of a TAP solid medium plate to obtain evenly distributed single colonies. The streaked plates were sealed and incubated at 25 °C under a 16:8 h light–dark cycle for approximately 5–7 days, until distinct, isolated green colonies were observed in the streaked area.

2. Plasmid Design, Synthesis, DNA Extraction, and Purification

To overexpress the endogenous mitochondrial chaperone protein CPN60C in

Chlamydomonas reinhardtii, we designed and constructed three plasmids (hereafter referred to as Plasmid A, Plasmid B, and Plasmid C). Each plasmid followed a core design concept: an expression cassette consisting of a validated C. reinhardtii promoter, endogenous gene, and terminator, inserted into a backbone widely used in transgenic Chlamydomonas research, and supplemented with paromomycin or hygromycin resistance markers.

Initially, we discovered that plasmid backbones commonly used in Chlamydomonas research were difficult to obtain domestically in China. International procurement was not feasible within the competition timeframe due to restrictions and uncertainties around microorganism importation. Full-sequence synthesis was considered, but it posed challenges of time, risk, and cost.

As a solution, we streamlined the original design, reducing its total length (including the backbone) to 6,928bp and attempting full-sequence synthesis, which refers to Plasmid C. (Due to synthesis delays, this plasmid was not completed within the competition timeframe; instead, its expression probability was inferred through statistical modeling.) Meanwhile, we contacted academic groups and institutions in China conducting Chlamydomonas research, and with their generous support, we obtained plasmid backbones that enabled the successful construction of Plasmid A and Plasmid B.

Plasmid A:
PsaD promoter + CPN60C CDS + PsaD terminator, inserted into a Chlamydomonas-compatible backbone containing a hygromycin resistance marker. This backbone (containing the promoter and terminator) was donated by Dr. Lei Zhao and Dr. Yuyong Hou’s team at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. Dr. Hou further assisted in optimizing the design and constructing the plasmid. Plasmid A was transformed into competent E. coli DH5α cells via the heat shock method.

Plasmid Extraction: Cells were harvested, resuspended in Solution I (suspension buffer with RNase A), lysed with Solution II (alkaline lysis buffer), and neutralized with Solution III. The supernatant was transferred to an adsorption column, washed with buffer to remove impurities, and eluted with sterile elution buffer to obtain highly purified plasmid DNA.

Plasmid B:
HSP70A–RBCS2 fusion promoter + CPN60C CDS + RBCS2 terminator, inserted into a C. reinhardtii backbone containing a paromomycin resistance marker. The backbone (including the target promoter and terminator with slightly different sequences) was donated by Dr. Kaiyao Huang and Dr. Lian Ye’s team at the Algae and Biomanufacturing Research Center, Institute of Hydrobiology, Chinese Academy of Sciences. After further optimization of our team, the plasmid was chemically synthesized and verified by Genewiz, and provided to us as sterile lyophilized plasmid powder.

3. Electroporation Transformation of Chlamydomonas reinhardtii

Cell Preparation:
A 6 mL culture of Chlamydomonas reinhardtii (UVM-4 or CC-124) in the mid-logarithmic growth phase (OD₇₅₀ ≈ 0.4–0.8) was harvested. Cells were washed twice by centrifugation (4,000 × g, 4 min, 4 °C) with ice-cold TAP medium containing 2% (w/v) sorbitol to remove conductive ions and maintain osmotic balance. The cells were then resuspended in 200 μL of TAP medium containing 2% sorbitol and incubated on ice for 10 minutes.

Electroporation:
A total of 120 μL of concentrated algal suspension was mixed with 20 μL of plasmid DNA (approximately 1–2 μg dissolved in sterile water) in a pre-chilled sterile electroporation cuvette (0.2 cm electrode gap). Electroporation was carried out on ice using a Bio-Rad Gene Pulser Xcell system with the following parameters: 2.6 kV and 400 Ω, corresponding to a pulse duration of approximately 0.1 ms as indicated by the instrument. Immediately after pulsing, 1 mL of TAP medium containing 2% sorbitol was added to the cuvette, and the mixture was gently transferred into a sterile centrifuge tube.

Recovery:
The transformed cells were supplemented with 10 mL of TAP medium containing 2% sorbitol and incubated statically at 25 °C under low light (approximately 5–10 μmol photons m⁻² s⁻¹) for 24 hours to promote recovery and allow expression of resistance genes.

4. Resistance Screening of Transformants

Preparation of Screening Plates:
To determine the optimal selection pressure, hygromycin (for Plasmid A) or paromomycin (for Plasmid B) was added to autoclaved TAP solid medium cooled to approximately 50 °C to achieve final concentrations of 5, 10, and 20 μg/mL, respectively. The medium was mixed thoroughly before pouring into plates (Berthold, Schmitt, & Mages, 2002; Biology LibreTexts, n.d.; Sizova, Fuhrmann & Hegemann, 2001).

Plating and Cultivation:
After 24 hours of dark recovery, 100 μL of the electroporated algal culture (Yamano et al., 2013) was gently mixed and evenly spread onto selection plates containing the corresponding antibiotic. As a negative control, an equal volume of untransformed algal culture was plated. All plates were inverted and incubated at 25 °C under a 16 h light / 8 h dark cycle (light intensity approximately 50–100 μmol photons m⁻² s⁻¹) for 7–10 days until distinct resistant transformant colonies appeared.

5. Screening and Verification of Positive Transformants

Selection and Expansion of Resistant Colonies:
Single, independent resistant colonies were picked from primary selection plates containing antibiotics and inoculated into fresh liquid TAP medium without antibiotics. Cultures were incubated statically under standard conditions (25 °C, 16 h light / 8 h dark cycle) until reaching the mid-logarithmic growth phase.

Sample Allocation and Clonal Preservation:
The expanded culture was divided into three portions. One portion was used for PCR verification (qualitative); one portion was used for qPCR verification (quantitative), while the other was streaked onto fresh TAP agar plates for temporary preservation of candidate positive clones.

Colony PCR Verification:

After a single algal colony has grown on a TAP plate, select an appropriate colony and culture it in liquid TAP. Once the culture reaches the logarithmic phase, carry out pretreatment. Add 10 μL of algal culture to 50 μL of 10 mM EDTA-Na₂ (pH 8.5), mix thoroughly, and incubate in a PCR machine at 100°C for 20 minutes. After treatment, centrifuge at 12,000 rpm for 2 minutes. Use 1 μL of the supernatant as the template for colony PCR to preliminarily verify plasmid transformation.

Note: Annealing temperature was set according to Tm ± 5°C.

Genomic DNA Extraction:
Total genomic DNA was extracted from presumptive positive colonies and wild-type control strains (negative control) using a Plant Genomic DNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing, China), following the manufacturer’s instructions. DNA concentration and purity (A260/A280) were measured with a NanoDrop spectrophotometer, and all samples were standardized to 20 ng/μL for use as qPCR templates.

qPCR Reaction:
Quantitative PCR was performed using SYBR Green qPCR premix (2× AccQ qPCR SYBR Green Master Mix; Accurate Biotechnology, China) on a Bio-Rad CFX96 real-time PCR system. Each 20 μL reaction contained 1× SYBR Green Master Mix, 0.2 μM of each forward and reverse primer, and approximately 50 ng of genomic DNA template.

Primers and Program:
Primers were specifically designed and synthesized for both the CPN60C gene and a housekeeping reference gene in Chlamydomonas reinhardtii. The qPCR program was as follows: initial denaturation at 95 °C for 5 min; followed by 40 cycles of denaturation at 95 °C for 15 s, annealing/extension at 60 °C for 30 s. A melting curve analysis was performed at the end of the reaction to verify product specificity.

Data Analysis:
Relative expression levels or copy numbers of CPN60C were calculated using the 2^(-ΔΔCt) method, normalized to the reference gene. Colonies showing significantly lower Ct values for the target gene compared with the wild-type control were identified as positive transformants.

Note: Annealing temperature was set according to Tm ± 5°C.

6. Heat Stress Treatment and Growth Phenotypic Analysis

Algal Culture:
Untransformed wild-type CC-124 and verified positive transformants were cultured in TAP liquid medium under standard conditions (25 °C, 16 h light / 8 h dark cycle) until early logarithmic growth (OD₇₅₀ ≈ 0.3). Subsequently, 10 mL of each culture was inoculated into 100 mL of fresh TAP medium in 150 mL Erlenmeyer flasks (inoculation ratio 1:10). This part of the experiment was carried out in Professor Zhen Wu’s laboratory at the Chongqing Institute of Traditional Chinese Medicine.

Heat Stress Treatment:
After inoculation, cultures were grown under standard conditions for three days, after which heat stress was applied. Entire Erlenmeyer flasks were immersed in water initially heated to 40 °C, then placed in a room-temperature (25 °C) water bath for 20 minutes. During this period, the water temperature gradually decreased from 40 °C, simulating a gradual heat stress process. After treatment, the flasks were removed, wiped dry, and immediately returned to standard culture conditions for recovery.

Growth Monitoring:
Due to limitations in experimental conditions at later stages, we were unable to perform daily destructive optical density (OD) measurements during the project recording period. Therefore, we continued culturing the negative control algal strains and transformants with different overexpression levels from a fixed growth starting point. Observational measurements were conducted by examining chlorosis (cell death) within the algal colonies, allowing us to qualitatively assess algal growth dynamics and tolerance to heat stress.

References

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