iGEM 2025 — Team shsd

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On this page

* [Part I – Chronological Notes](#part-1)
* [Part II – Handwritten Notes](#part-2)

## Part I – Chronological Notes

## Part II – Handwritten Notes

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On this page

* [Overview](#overview)
* [Case A · The Construction of Plasmids](#case-a)
* [Case B · The Construction of an Algae Incubator](#case-b)
* [— Cycle 1](#cycle-1)
* [— Cycle 2](#cycle-2)
* [— Cycle 3](#cycle-3)
* [References](#references)

## Overview

Engineering Success is a core component of the iGEM competition, encouraging teams to apply engineering thinking and strategies to address practical problems both within and beyond their projects. Throughout our months of work, we repeatedly went through the engineering cycle—Design → Build → Test → Learn—as we constructed and refined solutions. In this section, we share two case studies that highlight our team’s experiences and insights in achieving engineering success.

## Case A: The Construction of Plasmids

The theme of our project was the construction of a plasmid containing the endogenous CPN60C gene from *Chlamydomonas reinhardtii*. This plasmid was then transformed into new strains of *C. reinhardtii* that overexpressed CPN60C, thereby enhancing their heat stress tolerance, so that maintaining their photosynthetic function under high temperature. This improvement promoted more effective carbon dioxide fixation through the improved survivability, contributing to the exploration of a green and environmentally friendly solution to the global warming crisis. The successful construction of the CPN60C plasmid was the cornerstone of our project’s continued development.

Our project also underwent a small shift in focus. Initially, we attempted to target the endogenous gene LCIB, but later identified CPN60C as a more suitable target. This adjustment did not affect the plasmid construction process or the engineering cycle described below, so it will not be elaborated on separately.

The overall engineering process for Case A is summarized in the figure below:

![Case A process](https://static.igem.wiki/teams/5732/images/engineering/0-process-1.webp)

### Design – Round 1: Free Construction of a Plasmid Containing the CPN60C Gene

Our team began with a clear strategy: to construct a functional CPN60C expression cassette by combining a well-established promoter and terminator with the endogenous CPN60C gene, and then inserting this cassette into a plasmid backbone that had been extensively validated in previous research and proven suitable for expression in *Chlamydomonas reinhardtii*.

Following the instruction of our advisor, Dr. Brace, the students first brainstormed and drafted a preliminary plasmid design based on literature research. Dr. Brace then refined and optimized this draft. The final plasmid design included the PsaD promoter, CPN60C CDS, and RBCS2 terminator, which together formed a functional expression cassette inserted into the pOpt\_Clover\_Paro plasmid backbone.

The complete plasmid map is shown below:

![Plasmid map](https://static.igem.wiki/teams/5732/images/engineering/1-popt-cpn60c-final-construct.webp)

Of which, the PsaD promoter and RBCS2 terminator are both widely used in *Chlamydomonas reinhardtii* research. Their high efficiency in transgenic experiments has been well demonstrated. The CPN60C CDS sequence was obtained from the Phytozome database (JGI, n.d.). Since it is an endogenous gene, codon optimization was not required.

### Build – Round 1: Scarce Backbone, Excessively Long Sequence

However, the design above quickly ran into unexpected difficulties during the construction phase. We knew that the pOpt\_Clover\_Paro plasmid backbone was widely used in *Chlamydomonas reinhardtii* and could be purchased from the Chlamydomonas Resource Center in the United States (ChlamyCollection, n.d.). We therefore assumed it would be equally easy to obtain in China. In reality, this plasmid backbone is a niche product with no public procurement channels in China. Moreover, our lab conditions and the technical expertise of our high school team were not sufficient for us to synthesize such a long plasmid backbone ourselves. Purchasing it from the US was also not feasible due to the policy constraints of Chinese customs on the importation of microorganisms. The delivery time from the US to our lab was completely unpredictable. When we contacted the Chlamydomonas Resource Center, we were told it might take weeks, months, or even longer.

We then reached out to the gene synthesis companies to help us synthesize the entire sequence, including the backbone. The feedbacks were discouraging: given the 11,763-bp length of the plasmid, the risk of failure was significant. Commercial companies estimated a synthesis cycle of more than two months (in reality, likely much longer), and the cost was far beyond our budget.

As a result, we abandoned our initial plan and entered the second round of design.

### Design – Round 2: Shortening the Sequence, Supported by Scholars

The central goal of the second round was to minimize the total sequence length while retaining a functional expression cassette. To achieve this, we:

* Replaced the promoter and terminator with the shorter HSP70A–RBCS2 fusion promoter and RBCS2 terminator from the iGEM Registry (shorter than the traditional versions).
* Switched the backbone to pKS-aphVIII-lox, a widely used *C. reinhardtii* plasmid backbone with paromomycin resistance and a shorter sequence.

This reduced the full plasmid length to 6,928 base pairs, with an estimated synthesis time of 5–6 weeks, it was more affordable within our budget and project timeline. We designated this design as Plasmid C.

At the same time, we considered another path: reaching out to domestic scholars. Since many labs in China conduct *C. reinhardtii* research, perhaps they already had the plasmid backbones we needed. Fortunately, two groups responded generously:

* Dr. Lei Zhao and Dr. Yuyong Hou from the Tianjin Institute of Industrial Biotechnology, China Academy of Science.
* Dr. Kaiyao Huang and Dr. Lian Ye from the Algae and Biomanufacturing Research Center, Institute of Hydrobiology, China Academy of Science.

Thanks to their help, we ended up with three plasmid designs:

**Plasmid A:** PsaD promoter + CPN60C CDS + PsaD terminator, embedded in a backbone with a hygromycin resistance cassette. Dr. Hou donated the backbone, optimized the expression cassette design, the promoter, and the terminator, and assisted us to construct the plasmid.

![Plasmid A map](https://static.igem.wiki/teams/5732/images/engineering/3-cassatea.avif)

**Plasmid B:** HSP70A–RBCS2 promoter + CPN60C CDS + RBCS2 terminator, embedded in a backbone with a paromomycin resistance cassette, donated and optimized by Dr. Huang and Lian Ye’s team; constructed via insertion by Genewiz.

![Plasmid B map](https://static.igem.wiki/teams/5732/images/engineering/4-cassateb.avif)

**Plasmid C:** HSP70A–RBCS2 promoter + CPN60C CDS + RBCS2 terminator, embedded in the pKS-aphVIII-lox backbone with paromomycin resistance; which should be fully synthesized by Tsingke Biotechnology.

![Plasmid C map](https://static.igem.wiki/teams/5732/images/engineering/2-cpn60c-cassette-in-pks-aphviii-lox-map.webp)

In the following text, we refer to these as Plasmid A, Plasmid B, and Plasmid C.

### Build – Round 2: Good and Bad News

With the generous support of researchers, plasmid construction was no longer a nightmare.

Plasmid A was constructed with the help of Dr. Hou.

Plasmid B was built using the backbone donated by Dr. Huang and Ye.

However, our original design, Plasmid C, remained problematic. Although its length had been shortened to under 7,000 base pairs, full-sequence synthesis still proved too challenging. Despite repeated attempts, including splitting them into fragments, low-temperature assembly, re-splitting, and changing competent cells, the synthesis could not be completed within our experimental timeline.

### Testing: Wet Lab vs. Dry Lab

After this build phase, with both successes and setbacks, we had Plasmids A and B successfully constructed, while Plasmid C failed to be built but still provided insights. We adopted different testing strategies:

* For Plasmids A and B, we moved to the wet lab (see our “Experiments” page). After several weeks of lab work, we successfully transformed wild-type *C. reinhardtii* CC-124 and the lab strain UVM4 with Plasmids A and B. Using resistance markers, we identified potential positive transformants, which were confirmed by PCR, qPCR, and heat stress growth assays.
* For Plasmid C, which failed to construct, we used modeling to predict the probability of successful CPN60C expression in *C. reinhardtii*. The results suggested that, if synthesized, the plasmid would likely succeed in expressing the gene. Details are provided on our “Model” page.

### Learn: Insights from Every Step

The “Learn” process has been one of comprehensive reflection. From design to build to testing, each stage gave us insights for improvement. We will apply these lessons in the next round of work and hope other teams can learn from our experience as well.

**Design phase:** It is essential to assess the team’s technical capabilities before designing plasmids and to confirm reliable sources for all components, especially larger backbones. Since our major exploration were focused on English-language literature, and our scientific advisor is US-based while our team is based in China, we overlooked key differences in available resources and processes. In the future, we will prioritize this. For Plasmids A, B, and C, we will also consider optimizations such as adding introns or adjusting codons to improve transcription and translation.

**Build phase:** Our experience shows that, given our limited technical skills, attempting to synthesize a long plasmid in its entirety is not a wise strategy in terms of time, cost, or likelihood of success. The risk of synthesis failure increases exponentially, not linearly, with sequence length.

**Test phase:** In testing Plasmids A and B, we identified areas for optimization in algal culture and expansion, electroporation transformation, resistance screening, and PCR. For example:

* *C. reinhardtii* has strict requirements for light, temperature, and circadian rhythm, which must be flexibly adjusted.
* Preliminary experiments, such as confirming antibiotic concentrations, help prevent wasted resources.
* Aseptic techniques are crucial throughout, especially for strain propagation, electroporation, and plating.

Finally, since CPN60C is a mitochondrial chaperone protein that consumes ATP, it may burden algal growth under non-stress conditions. This calls for a more conservative preservation strategy for electroporated strains, particularly UVM4 strains with cell wall defects.

## Case B: The Construction of an Algae Incubator

*Chlamydomonas reinhardtii* is highly sensitive to light, temperature, and circadian rhythms. Compared to many other plants, it is also relatively fragile under laboratory conditions. As a result, cultivating algae became the very first challenge in our early lab work.

Ideally, cultivating *C. reinhardtii* requires an incubator with built-in, controllable lighting. However, such incubators are costly and have limited applications. Our lab did not have this equipment, and purchasing additional devices was beyond our team’s budget. Therefore, we adapted to our local circumstances and set out to construct a custom-made incubator with controllable lighting.The overall engineering process for Case B is summarized in the figure below:

![Case B process](https://static.igem.wiki/teams/5732/images/engineering/01-process-2.webp)

## Cycle 1

### Design: Full-Spectrum LED Light + Traditional Incubator

The core of the design was to adapt a traditional incubator to provide controllable lighting suitable for the growth of *Chlamydomonas reinhardtii*. Starting from first principles, we quickly settled on a design solution: adding a suitable incubation lamp to the traditional incubator.

After researching commercially available lamps, we selected a full-spectrum LED desk lamp with both red and blue wavelengths. Its full-spectrum light made it suitable for growing *C. reinhardtii*. As a cold light source, LEDs do not generate excess heat that might affect algae growth. Its size allowed it to fit inside the incubator while providing relatively even illumination throughout. We also added an external rotary switch to control light intensity and circadian rhythm without disturbing the algae’s growth.

### Build: First Version of the Homemade Illuminated Incubator

The process of building our custom illuminated incubator went smoothly. The full-spectrum LED desk lamp fit the incubator’s dimensions perfectly and provided uniform illumination inside. The external rotary switch also worked as expected. Overall, the setup met our design goals.

![Homemade illuminated incubator v1](https://static.igem.wiki/teams/5732/images/engineering/7-illumination-incubator.webp)

### Test: First Batch of Algae Cultures and Unexpected Temperature Issues

We began cultivating our first batch of algae in the custom incubator. For the first few days, both light and temperature were well controlled, and algae expansion proceeded smoothly.

Soon, however, we encountered an unexpected problem: extremely hot weather.

The incubator has a built-in temperature control system that normally maintains a constant 25°C, so we had not considered temperature as a major factor in the initial design. But since our experiments took place during the summer, with outdoor temperatures exceeding 40°C (104°F), the incubator’s cooling system was under greater strain. After a few days, the system nearly failed. The temperature inside the incubator rose from 25°C to 27°C, and eventually reached 31°C—essentially a “sauna” for the fragile algae.

![Too hot](https://static.igem.wiki/teams/5732/images/engineering/8-too-hot.webp)

### Learn: Incorporating Temperature Control into the Second Round of Design

The results of the first round, along with this unexpected issue, forced us to rethink our design: we had to account for temperature control. We quickly began developing a new solution.

## Cycle 2

### Design: A “Refrigerator” Illuminated by Full-Spectrum LED Lighting

Our second-round design again followed first principles: meeting the light, circadian rhythm, and temperature requirements for *C. reinhardtii* was the sole starting point. To address the temperature runaway issue, we devised a simple and cost-effective solution: placing an open rectangular container of ice cubes at the bottom of the incubator. This provided additional cooling and reduced the load on the incubator’s own temperature control system.

### Build: Second Version of the Homemade Illuminated Incubator

Like the first version, construction posed no particular difficulties. Through several trials, we adjusted the amount of ice, eventually determining the right volume to support the incubator’s temperature control system without interfering with algae growth.

### Test: New Issues with Manual Temperature Control

We resumed cultivating *C. reinhardtii* in the second version of the incubator. This time, the temperature issue was resolved. With the help of the ice, the incubator maintained a stable 25°C, and the algae grew normally for several days.

However, this manual cooling method introduced new problems. Because of the persistent summer heat, the ice had to be replaced every few hours. This not only made the experiments more tedious, but also caused temperature and light fluctuations each time the incubator door was opened, especially difficult to manage at night.

### Learn: Continuing to Solve New Problems

After the second engineering cycle, we entered a new stage of reflection. Using ice to cool the incubator solved one problem but created another: the need for constant manual intervention.

At this point, we began to ask ourselves: Are we limiting our thinking by being tied to the “box” of the incubator? Must everything—from temperature to light control—be managed inside a single box? Or do we really need a box at all?

## Cycle 3

### Design: The Third Version of the “Homemade Illuminated Incubator”—But Not a “Box”

Building on the second engineering cycle, we returned to first principles and freed ourselves from the constraint of the “incubator” concept. What we truly needed was simply a small space that could reliably control light, temperature, and circadian rhythms, ensuring stable conditions for the growth of *Chlamydomonas reinhardtii*. Whether it was a box or something else no longer mattered.

We initially considered using a small darkroom, but preliminary research revealed that the air conditioner lacked proper drainage, and condensation could damage the microscope in the room. Ultimately, we settled on a small room (adjacent to the cell room) equipped with a central climate control system and heavy blackout curtains. This became our third version of the “homemade illuminated incubator.”

### Build: Special Arrangement for a Small Room

We specially configured the small room to meet the requirements of a “homemade light-incubator.” First, we repeatedly verified the performance of the room’s central thermostat, using a smart thermo-hygrometer and an app to monitor temperature fluctuations. Second, we added extra layers to the existing blackout curtains to ensure complete darkness during the night cycle. Finally, we placed the full-spectrum LED desk lamp we had originally purchased on the surface where algae cultures were kept, providing relatively even illumination for all strains.

![Small room setup](https://static.igem.wiki/teams/5732/images/engineering/9-the-room.webp)

### Test: Finally, We Can Cultivate Algae in Peace

The third round of testing went smoothly and ultimately became our final method for cultivating, expanding, and incubating algae strains. The small, improvised darkroom functioned as a large incubator and provided a solid foundation for our entire experimental process.

### Learn: Lessons

After three engineering cycles, we finally created a stable and usable homemade light-incubator, enabling the effective expansion of algae strains. At each round of improvement, we often solved one problem only to introduce another. Interestingly, this mirrors the biology of *Chlamydomonas reinhardtii*: many of its functions are tightly coupled, so improving one function may reduce the efficiency of another, strongly correlated function, leading to a negative overall effect. This was a small but striking example of synesthesia we experienced firsthand.

Nevertheless, the most important lesson from this engineering success story is the value of applying first principles and defining our goals clearly, rather than being constrained by existing options.

## References

- Chlamydomonas Resource Center. (n.d.). pOpt\_Clover\_Paro. Retrieved from https://www.chlamycollection.org/product/popt\_clover\_paro/

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On this page

* [1. Activation, Expansion, and Cultivation of *Chlamydomonas reinhardtii*](#section-1)
* [— 1.1 Activation and Cultivation of UVM-4](#section-1-1)
* [— 1.2 Propagation of CC-124](#section-1-2)
* [— 1.3 Plate Streaking Method](#section-1-3)
* [2. Plasmid Design, Synthesis, DNA Extraction, and Purification](#section-2)
* [3. Electroporation Transformation of *Chlamydomonas reinhardtii*](#section-3)
* [4. Resistance Screening of Transformants](#section-4)
* [5. Screening and Verification of Positive Transformants](#section-5)
* [6. Heat Stress Treatment and Growth Phenotypic Analysis](#section-6)
* [References](#references)

## 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.

![UVM-4 culture](https://static.igem.wiki/teams/5732/images/experiments/uvm4.webp)

### 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.

![CC-124 colonies](https://static.igem.wiki/teams/5732/images/experiments/cc124.webp)

## 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.

![Electroporation procedure](https://static.igem.wiki/teams/5732/images/experiments/electroporation.webp)

![Electroporator](https://static.igem.wiki/teams/5732/images/experiments/electroporator.webp)

## 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.

![PCR table 1](https://static.igem.wiki/teams/5732/images/experiments/table1.webp)

![PCR table 2](https://static.igem.wiki/teams/5732/images/experiments/table2.webp)

![PCR table 3](https://static.igem.wiki/teams/5732/images/experiments/table-3.webp)

*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.

![qPCR table 4](https://static.igem.wiki/teams/5732/images/experiments/table-4.webp)

![qPCR table 5](https://static.igem.wiki/teams/5732/images/experiments/table-5.webp)

*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

* Barolo, L., Commault, A. S., Abbriano, R. M., Padula, M. P., Kim, M., Kuzhiumparambil, U., ... & Pernice, M. (2022). Unassembled cell wall proteins form aggregates in the extracellular space of *Chlamydomonas reinhardtii* strain UVM4. *Applied Microbiology and Biotechnology*, 106(11), 4145-4156.
* Berthold, P., Schmitt, R., & Mages, W. (2002). An engineered *Streptomyces hygroscopicus* aph7″ gene mediates dominant resistance against hygromycin B in *Chlamydomonas reinhardtii*. *Protist*, 153(4), 401-412.
* Biology LibreTexts. (n.d.). Adding antibiotics. In Lab manual: Synthetic biology protocols. LibreTexts. Retrieved September 29, 2025, from https://bio.libretexts.org/Bookshelves/Biotechnology/Lab\_Manual%3A\_Synthetic\_Biology\_Protocols/03%3A\_Ancillary\_Materials/3.1.02%3A\_Adding\_Antibiotics
* Chioccioli, M., Hankamer, B., & Ross, I. L. (2014). Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae *Chlamydomonas reinhardtii* and *Chlorella vulgaris*. *PloS one*, 9(5), e97269.
* Chlamydomonas Resource Center. (n.d.). TAP and Tris-minimal. Chlamydomonas Resource Center. Retrieved September 29, 2025, from https://www.chlamycollection.org/methods/media-recipes/tap-and-tris-minimal/
* Corredor, L., Barnhart, E. P., Parker, A. E., Gerlach, R., & Fields, M. W. (2021). Effect of temperature, nitrate concentration, pH and bicarbonate addition on biomass and lipid accumulation in the sporulating green alga PW95. *Algal Research*, 53, 102148.
* Gao, K. (2021). Approaches and involved principles to control pH/pCO2 stability in algal cultures. *Journal of Applied Phycology*, 33(6), 3497-3505.
* Haire, T. C., Bell, C., Cutshaw, K., Swiger, B., Winkelmann, K., & Palmer, A. G. (2018). Robust microplate-based methods for culturing and in vivo phenotypic screening of *Chlamydomonas reinhardtii*. *Frontiers in plant science*, 9, 235.
* Harris, E. H. (2009). *The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use: Volume 1* (Vol. 1). Academic Press.
* Kumar, S. V., Misquitta, R. W., Reddy, V. S., Rao, B. J., & Rajam, M. V. (2004). Genetic transformation of the green alga—*Chlamydomonas reinhardtii* by *Agrobacterium tumefaciens*. *Plant Science*, 166(3), 731-738.
* Neupert, J., Karcher, D., & Bock, R. (2009). Generation of *Chlamydomonas* strains that efficiently express nuclear transgenes. *The Plant Journal*, 57(6), 1140-1150.
* Sizova, I., Fuhrmann, M., & Hegemann, P. (2001). A *Streptomyces rimosus* aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to *Chlamydomonas reinhardtii*. *Gene*, 277(1-2), 221-229.
* Yamano, T., Iguchi, H., & Fukuzawa, H. (2013). Rapid transformation of *Chlamydomonas reinhardtii* without cell-wall removal. *Journal of Bioscience and Bioengineering*, 115(6), 691-694.

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On this page

* [Abstract](#abstract)
* [1. Antibiotic Sensitivity Assay](#section-1)
* [2. Optimal Resistance Screening Concentration](#section-2)
* [3. Effect of Algal Strain Genetic Background on Transformation Efficiency](#section-3)
* [4. Effect of Strain Genetic Background on Viability](#section-4)
* [— 4.1 Cultivation of Negative Control Strain](#section-4-1)
* [— 4.2 Cultivation of Transformed Strains](#section-4-2)
* [5. PCR Verification of Transformed CC-124 Algae](#section-5)
* [6. qPCR Verification of Transformed CC-124 Algae](#section-6)
* [7. Heat Stress Experiment Results](#section-7)
* [8. Experimental Shortcomings and Next Steps](#section-8)
* [References](#references)

## 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).

![Fig. 1](https://static.igem.wiki/teams/5732/images/results/fig-1.webp)

Fig. 1 Growth of Chlamydomonas reinhardtii UVM-4 transformed with plasmid A on plates containing different concentrations of hygromycin

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.

![Fig. 2](https://static.igem.wiki/teams/5732/images/results/fig-2.webp)

Fig. 2 Growth of UVM-4 and CC-124 algal colonies on plates after electroporation

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.

![Fig. 3](https://static.igem.wiki/teams/5732/images/results/fig-3.webp)

Fig. 3. Growth and passage of UVM-4 (Negative) and CC-124 (WT) strains.

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

![Fig. 4](https://static.igem.wiki/teams/5732/images/results/fig-4.webp)

Fig. 4. Propagation culture of presumed positive algal colonies after transformation
(A: UVM-4 (negative); B & C: UVM-4 CPN60C+; D: CC-124 WT; E & F: CC-124 CPN60C+)

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

![Fig. 5](https://static.igem.wiki/teams/5732/images/results/fig-5.webp)

Fig. 5 PCR verification of CPN60C-overexpressing algae
(Note: M: marker D2000; Lanes 1–3: CC-124 transformed with plasmid A; Lanes 4–6: CC-124 transformed with plasmid B)

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

![Fig. 6](https://static.igem.wiki/teams/5732/images/results/fig-6.webp)

Fig. 6. Relative expression levels of the CPN60C gene in the transformed CC-124 strain

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).

![Fig. 7](https://static.igem.wiki/teams/5732/images/results/fig-7.webp)

Figure 7. Overexpression of CPN60C Improves Heat Tolerance in *Chlamydomonas reinhardtii*
(Note:
CPN60C+-1 and CPN60C+-2 represent CC-124 strains transformed with plasmids A and B carrying the CPN60C gene, respectively.
1. In the first row, the photos of each sample were taken separately and then arranged in a column, which results in differences in brightness. The second row was taken under the same lighting conditions to better show the differences in their degree of chlorosis.
2. The samples in the two rows correspond vertically. The rubber band at the seal of the rightmost sample in the second row is a different color from that in the first row, which is because we once attempted to open the wrap daily to measure the pH value. Later, as the experimental conditions could not provide a reasonable method of measurement, this attempt was suspended. During the process, the rubber band of the rightmost sample broke and was therefore replaced with a new one.
3. In the second row, the scale on the conical flask of the middle sample is blocked by the wrap.
)

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

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