iGEM 2025 — Team kcis-xiugang-taipei

2024 FALL
2024 WINTER
2025 SPRING
2025 SUMMER

* [Inspiration](#inspiration)
* [Our Journey](#journey)
* [Applications](#application)
* [Validation](#validation)
* [References](#references)

![Figure 1](https://static.igem.wiki/teams/5624/engineering/engin-fig-1.webp)

Figure 1: iGEM Engineering Cycle

### What inspired our team to initiate this project?

Currently, one of the most prevalent artificial carbon sequestration methods is Carbon Capture and Storage (CCS), a costly and energy-intensive technology with limited efficacy. As a result, the team resorts to biological carbon fixation approaches, planning on exploiting the dominant photosynthetic method via oxygenic photosynthesis. In fact, according to the Encyclopedia of the Environment, photosynthesis solely contributes to approximately 115 to 120 gigatons of carbon fixation annually from CO2 in the atmosphere (Jean-François, 2025). The significance of such biochemical processes inspired our team to leverage its efficiency for carbon sequestration.

### Why did we choose to use cyanobacteria?

#### Photorespiration – A Fatal Drawback

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO), a key enzyme that takes part in the Calvin cycle through binding with atmospheric CO2, struggles with the lack of substrate specificity and slow product turnover rates. This leads to the enzyme fixing CO2 with a low efficiency. RuBisCO doesn’t only work for CO2. It also binds with oxygen through a metabolic process known as photorespiration (Cummins et al., 2018). Photorespiration results in the loss of 3 fixed carbons per 3 turns of the Calvin cycle and is therefore undesirable, as it does not contribute to carbon fixation; instead, it wastes oxygen and energy.

#### Inspirations from Alternative Photosynthetic Pathways

To counter RuBisCO’s low affinity to carbon and minimize the effects of photorespiration in harsh, arid, and dry environments, two adaptive pathways, C4 and CAM photosynthesis, emerged among plants to optimize carbon fixation while conserving water. C4 photosynthesis is a two-step process that completes carbon fixation at two distinct locations: the mesophyll and bundle sheath cells. On the other hand, CAM photosynthesis also involves two stages, yet within the same cell at different times (C4 and CAM Photosynthesis | Research Starters | EBSCO Research, n.d.).

C4 and CAM photosynthesis, evolutionary strategies that address the low carbon fixation rates in traditional C3 photosynthesis, provide the team with insights into modulating the biological pathways in photosynthetic organisms to facilitate carbon fixation and mitigate the adverse impacts of industrial carbon emissions more effectively.

### How can we use cyanobacteria to address photorespiration?

Cyanobacteria, also known as blue-green bacteria and blue-green algae, is a phylum of bacteria that perform oxygenic photosynthesis. In particular, the CCM in cyanobacteria is considered a model for improving photosynthesis in C3 plants. Thus, combating the drawbacks associated with photorespiration, as mentioned, cyanobacteria become a promising organism that could be used. Furthermore, cyanobacteria are integral primary producers. Currently, an estimated 20-30% of the Earth’s organic carbon is derived from cyanobacterial carbon fixation (Zhou et al., 2016).

Given that increasing RuBisCO’s affinity to CO2 in the CCM directly optimizes the Calvin cycle, the team proposes the genetic modification of the freshwater cyanobacterial strain Synechococcus elongatus PCC 7942 by modifying the expression of a HCO3- transporter BCT1 of the carbon uptake system. By increasing the expression of the *cmpABCD* operon, which utilizes light-inducible promoters to enhance the amount of BCT1 on the cyanobacterial inner membrane, the team aimed to increase the concentration of CO2 in the carboxysomes, thereby upregulating RuBisCO activity and improving carbon fixation efficacy while reducing photorespiration. When CO2 is concentrated and abundant, RuBisCO preferably binds to CO2 rather than O2. This is because the binding of CO2 and O2 to RuBisCO’s binding site is a competitive process, so under high CO2 levels, CO2 outcompetes O2 in terms of binding and promotes the carboxylation reaction over the oxygenation reaction.

### What are some concerns with the utilization of the *cmpAB*CD Operon?

One of the problems our team encountered was the size of the *cmpABCD* operon, which spans about 5000 base pairs. Cloning large DNA sequences is technically challenging due to insufficient ligation and the difficulties associated with transformation. Therefore, to overcome this difficulty, we adopted a modular design by splitting the operon into two transcriptional units: *cmpAB* and *cmpCD*. This separation enables more efficient cloning and theoretically lowers error and mutation rates.

### Why did the team decide to use only the psbA1 promoter rather than both the psbA1 and psbII promoters when constructing the plasmids?

While conducting synthetic biology experiments aimed at constructing the psbA1-*cmpAB* and psbII-*cmpCD* constructs, the team encountered difficulties with assembling the psbA1-*cmpAB* construct. To address this issue, the team conducted a thorough analysis of each procedural step, starting with sequence alignments. This detailed investigation uncovered that the pAM1619 recombinant plasmid, sourced from Addgene and purported to contain the psbA1 promoter, did not carry the correct psbA1 promoter sequence. Additionally, the pCOTS-pyl-GFP(35TAG) plasmid, also obtained from Addgene and labeled as containing the psbII promoter, was found to harbor the psbA1 promoter sequence instead. In fact, the team decided to generate psbA1-*cmpAB* and psbA1-*cmpCD* on the pCOTS-pyl-GFP(35TAG) plasmid, respectively.

### How did our team evaluate the potential advantages of creating *cmpAB* and *cmpCD* fusion proteins?

To justify the assembly and stability of the *cmpAB* and *cmpCD* fusion genes, the team utilized AlphaFold and DeepTMHMM to predict the 3D protein structures and orientations of the *cmpAB* and *cmpCD* fusion proteins using their amino acid sequences and then compared the predicted configurations to the assembly of individual *cmpA*, B, C, and D compartments. Through qualitative protein structural visualization and quantitative analyses, including root mean square deviation values, predicted topology plots, and signal peptide charts, the team concluded that the *cmpAB* and CD fusion proteins demonstrate local structural similarity and a coinciding orientation compared to the individual *cmpA*, B, C, and D compartments. The detailed results can be found under the “Protein Modeling” section. After proving the stability of the *cmpABCD* (BCT1) complex when split into *cmpAB* and *cmpCD* fusion proteins, we moved on to construct the genetic parts—*cmpAB* and *cmpCD* fusion genes—that included the light-dependent promoter ppsbA1 in the expression vectors to drive the constructs’ overexpression.

![Figure 2](https://static.igem.wiki/teams/5624/engineering/figure-2.avif)

Figure 2: BCT1 Gene Strategy Cycle

### What challenges did our team face when conducting the mathematical modeling of the CCM?

One of the significant challenges our team encountered when attempting to model the CCM mathematically was our lack of prior experience in computational biology. Initially, we knew very little about how to develop a plausible mechanism. In fact, we acquired some basic knowledge, such as enzyme kinetics and MATLAB coding, from our advisors. Subsequently, we sought advice from Prof. Mangan at Northwestern University, who specializes in CCM modeling, after we found and read her paper “Systems analysis of the CO2 concentrating mechanism in cyanobacteria.” (Mangan & Brenner, 2014) During our consultation, Prof. Mangan explained that efficiency in computational models is not solely defined by RuBisCO activity, but also depends heavily on auxiliary components such as carbonic anhydrase and the structural/functional constraints of carboxysomes. Following her guidance, we incorporated her CCM diagram logic into our MATLAB simulations.

Moreover, since experimental measurements of metabolite concentrations and enzyme kinetics in cyanobacteria are often incomplete or inconsistent, we were forced to build from scratch. For example, to simplify diffusion dynamics, we assumed the outer membrane, inner membrane, and carboxysome to be perfect concentric spheres. This idealization allowed us to approximate how CO2 would diffuse inward and be trapped by the carboxysome.

![Figure 3](https://static.igem.wiki/teams/5624/engineering/figure-3.webp)

Figure 3: CCM Modeling Cycle

### How did our team successfully overcome initial obstacles to grow cyanobacteria?

Upon receiving a plate of *S. elongatus*  PCC 7942 from Dr. Lan at National Yang-Ming Chiao-Tung University, we grew the cells in a flask containing 100 mL of BG-11. After not observing any growth for weeks (BG-11 liquid cultures were transparent in color weeks after inoculation), we attributed the failure to the low initial cell density of cyanobacteria, as it impeded the cells’ multiplication and growth. To combat this, we switched to smaller 5 mL liquid cultures to increase the cell density.

Additionally, since our newly streaked plates did not yield enough cells to collect with an inoculation loop, we had to transfer entire agar chunks into 15 mL conical tubes to obtain sufficient biomass. For about a month, all our cultures contained agar pieces, which made it difficult to scale up because pipette tips frequently collided with the agar during liquid transfers.

Eventually, we observed growth in the 15 mL tubes, visible as green, cloudy sediment at the bottom and small bubbles rising along the surface. These signs of respiration confirmed that the cyanobacteria were alive and metabolically active. We then transferred the cultures into 100 mL flasks and diluted them with BG-11 medium, which supported faster growth.

In conclusion, to grow cyanobacteria successfully, start with small liquid cultures inoculated from colonies, and then expand them stepwise into larger volumes to ensure healthy growth of the cyanobacteria.

![Figure 4](https://static.igem.wiki/teams/5624/engineering/figure-4.webp)

Figure 4: Growth Optimization Cycle

### What challenges did our team face on plasmid transformation, and what methods did we try?

Cyanobacterial transformation has been one of the main challenges we encountered throughout our project. We have applied various transformation methods, including chemical transformations, natural transformation, and electroporation, over several months. But no colonies were observed throughout these attempts. On our last attempt at using natural transformation, colonies were observed, but the plates were contaminated, making it impossible to determine the success of the transformation.

With the consistent failure, the team contacted Dr. Hsuan-Chen Wu, associate professor at National Taiwan University. Through several discussions, he informed the team that, besides contamination, the constant failure of transformation can be attributed to the use of aged cyanobacteria. Our team relied on the initial one-plate Dr. Lan sent us for experiments over a period of nine months. Therefore, when we reached the transformation phase, the cyanobacteria were already several months old, making them very difficult to transform.

Older cyanobacterial cultures often enter the stationary or decline phase of growth, during which metabolic activity, membrane fluidity, and DNA uptake efficiency are reduced compared to cells in the exponential phase. During the stationary phase, nutrient depletion and oxidative stress can lead to the formation of thickened cell walls and changes in the extracellular polysaccharide (EPS) layer, both of which can act as physical barriers to DNA uptake. Additionally, competence in many bacteria is tightly regulated and usually peaks during active growth, meaning that aged cyanobacteria may have been physiologically less receptive to foreign DNA. Taken together, these factors could explain the low transformation efficiency observed (Berla et al., 2013).

![Figure 5](https://static.igem.wiki/teams/5624/engineering/figure-5.webp)

Figure 5: Transformation Pipeline Cycle

### How did we test the functionality of our engineered cyanobacteria?

To evaluate the effectiveness of our engineered cyanobacteria within a carbon fixation device, we conducted a CO2 uptake assay adopted from previously established protocols for cyanobacterial systems. With the assay performed on a cyanobacteria-containing experimental group using a BG-11 control group as a reference, we obtained key parameters, such as CO2 decreasing patterns and equilibrium concentrations, for the wild-type cyanobacterial strain *S. elongatus* PCC 7942. These data retrieved could then be employed in our cyanobacterial CCM mathematical model as a baseline to quantify the enhancement in CO2 uptake efficiency for our engineered strain, thereby confirming the functional overexpression of BCT1 transporters.

![Figure 6](https://static.igem.wiki/teams/5624/engineering/figure-6.avif)

Figure 6: Validation & Hardware Cycle

### Applications

Our cyanobacteria-powered bioreactor is designed for carbon capture and fixation in the industries that are categorized as scope 1 CO2 emitters. We anticipate its primary application will be at coal-fired and natural gas power plants, which are the primary point sources of CO2 emissions. The device can also be adapted for use at other industrial sites with high CO2 output, such as cement manufacturing facilities and steel mills. The bioreactor’s modular design allows it to be scaled to match the emission levels of the facility’s output.

The device will be implemented as a closed-loop system integrated into a power plant’s existing infrastructure. A portion of the flue gas, previously purified to remove other toxic particles, will be diverted into the bioreactor’s cultivation medium to serve as the essential carbon source for the cyanobacteria. Inside the bioreactor, the cyanobacteria will perform their natural biological processes, including photosynthesis, consuming the CO2 to produce biomass. Once the culture reaches a sufficient density, this biomass will be harvested and processed into animal feed, and the nutrient mediums will be replaced. By transforming a waste stream into a resource, our technology creates a circular economy model for the energy sector, providing a practical and economically viable solution to reduce greenhouse gas emissions while creating sustainable industries.

### How did we validate our ideas?

We validated our ideas by consulting specialists across materials science, microbiology, and computational modeling, who provided feedback that shaped our project into something both feasible and responsible. Since our project centered on enhancing carbon fixation in cyanobacteria, we also engaged with cyanobacteria researchers to test the biological feasibility of our approach. From these conversations, we learned several critical points. First, RuBisCO is already operating close to saturation under normal conditions, meaning that increasing carbon fixation is not as simple as overexpressing the enzyme. Second, the pore size of the carboxysome shell imposes diffusion limits on bicarbonate and CO2, reminding us that structural constraints must be considered in our design. Finally, we were informed that the cyanobacterial carbon concentrating mechanism (CCM) is only activated under low ambient CO2, which clarified the environmental conditions under which our modifications could realistically work. This expert feedback prompted us to shift focus from manipulating RuBisCO directly to improving bicarbonate transport and CCM efficiency.

To further ground our design in feasibility, we consulted with modeling specialists who helped us identify the variables that were most critical to simulate. They highlighted parameters such as bicarbonate transport rates, internal CO2 concentrations, and carboxysome permeability as key to predicting whether our design could succeed. Their input guided us in refining our models, enabling us to validate core assumptions before moving into the wet lab.

Through this iterative process, expert feedback reshaped our project direction. Instead of pursuing overly ambitious or biologically unrealistic approaches, we refined our design to align with the constraints of cyanobacterial metabolism and the opportunities within synthetic biology. These consultations not only validated our ideas but also ensured that our project was responsible, scientifically sound, and implementable.

## References

Berla, B. M., Saha, R., Immethun, C. M., Maranas, C. D., Moon, T. S., & Pakrasi, H. B. (2013). Synthetic Biology of cyanobacteria: Unique Challenges and opportunities. Frontiers in Microbiology, 4. https://doi.org/10.3389/fmicb.2013.00246

C4 and CAM photosynthesis | Research Starters | EBSCO Research. (n.d.). EBSCO. https://www.ebsco.com/research-starters/botany/c4-and-cam-photosynthesis

Cummins, P. L., Kannappan, B., & Gready, J. E. (2018). Directions for optimization of photosynthetic carbon fixation: RUBISCO’s efficiency may not be so constrained after all. Frontiers in Plant Science, 9. https://doi.org/10.3389/fpls.2018.00183

Jean-François, M. (2025, January 5). The path of carbon in photosynthesis - Encyclopedia of the Environment. Encyclopedia of the Environment. https://www.encyclopedie-environnement.org/en/life/path-carbon-photosynthesis-2/

Libretexts. (2024, November 23). 8.9A: Cyanobacteria. Biology LibreTexts. https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology\_(Boundless)/08%3A\_Microbial\_Evolution\_Phylogeny\_and\_Diversity/8.09%3A\_Nonproteobacteria\_Gram-Negative\_Bacteria/8.9A%3A\_Cyanobacteria

Mangan, N. M., & Brenner, M. P. (2014). Systems analysis of the CO2 concentrating mechanism in cyanobacteria. elife, 3, e02043

Payton, B. (n.d.). Industrial Transformation – TCAN 台灣氣候行動網絡. TCAN 台灣氣候行動網絡. Retrieved September 11, 2025, from https://tcan2050.org.tw/en/project/industrial-transformation-en/

Zhou, J., Zhu, T., Cai, Z., & Li, Y. (2016). From cyanochemicals to cyanofactories: a review and perspective. Microbial Cell Factories, 15(1). https://doi.org/10.1186/s12934-015-0405-3

Show all

* [Experimental Goal](#experimental-goal)
* [Gene Cloning](#gene-cloning)
* [Cyanobacteria Transformation](#cyanobacteria-transformation)
* [Functional Analyses](#funtional-analyses)
* [Expression Confirmation](#expression-confirmation)
* [Additional Experiments](#additional-experiments)
* [Protocols](#experiment-protocols)
* [References](#references)

### Experimental Goal

Our project focuses on genetically modifying cyanobacteria to enhance carbon fixation efficiency by boosting their native carbon-concentration mechanism (CCM). To specifically improve the efficiency of bicarbonate (HCO3-) uptake via the BCT1 transport protein, the team engineered two fusion genes, *cmpAB* and *cmpCD*, driven by the *ppsbA1* light-response promoter, rather than expressing each genetic component separately. After confirming the feasibility of our hypothetical design with institute professors, the team confirmed the functionalities of our *cmpAB* and *CD *fusion proteins, and that their expression could significantly enhance the cyanobacterial CCM pathway and carbon fixation rates. Ultimately, we aim to boost cyanobacterial photosynthetic efficiency under varying environmental carbon conditions.**

### Choosing the Target Genes

The carbon uptake mechanism in cyanobacteria is a complex system that can be divided into five distinct components, with three of these being HCO3- transporters located on the plasma membrane. One of the most critical transporters is HCO3- Transporter 1 (BCT1), an ATP-binding cassette (ABC) transporter encoded by the *cmpABCD* operon (Kupriyanova et al., 2023). This operon encodes a four-component complex consisting of polypeptide subunits:

1. *cmpA*: *cmpA* is a periplasmic solute-binding lipoprotein facing the cytoplasm, where it captures HCO3- through its specialized active site and delivers it to the transporter complex *cmpB*. Structural studies confirmed that *cmpA* has a high affinity and specificity for HCO3-, playing a crucial role in the ABC transporter system (Koropatkin et al., 2007).

2. *cmpB*: *cmpB* is a transmembrane protein that forms the homodimer channel, facilitating HCO3- to pass into the cytoplasm as part of the active HCO3- uptake system. However, *cmpB* alone cannot transport HCO3- against its concentration gradient; it needs the two ATPase subunits, *cmpC* and *cmpD*, to activate the HCO3- transport process. This system is vital for cyanobacteria to sufficiently uptake and concentrate inorganic carbon for photosynthesis under low CO2 conditions (Omata et al., 1999).

3. *cmpC*: *cmpC* is an ATPase/substrate-binding protein that plays a dual role in binding HCO3- and hydrolyzing ATP. *cmpC* hydrolyzes ATP through its N-terminal ATP-binding domain to supply the energy needed for HCO3- translocation into the cytoplasm, and regulates HCO3- transport through its C-terminal substrate-binding domain (Price et al., 2008).

4. *cmpD*: *cmpD* is a cytoplasmic ATPase that couples ATP hydrolysis to fuel HCO3- transport (Koropatkin et al., 2007). *cmpD*, working together with *cmpC*, forms the ATP-hydrolyzing motor to provide energy for the *cmpB* homodimeric channel, importing HCO3- into the cyanobacterial cytoplasm. This ATP-hydrolyzing motor powers active HCO3- transport, enabling cyanobacteria to concentrate inorganic carbon for photosynthesis under low CO2 conditions (Omata et al., 1999; Price et al., 2008).

Research by Omata et al. determined that the original *cmpABCD* operon is induced under low CO2 conditions (typically below 0.035% CO2, vol/vol), where it plays a vital role in enhancing the CCM by intaking HCO3- (Omata et al., 1999). Because the CCM works primarily under low CO2 levels, its efficiency for uptaking HCO3- is relatively low under high CO2 levels. Therefore, our team employs a light-inducible promoter to maintain the expression and efficiency of the *cmpABCD* genes under a wide range of CO2 levels, which is discussed in greater detail in the next section (Van der Staay et al., 1991; Nair et al., 2001).

### Fusion Gene Design and Verification

#### Plasmid Maps

Our team used the SnapGene software to design the ppsbA1-*cmpAB* and ppsbA1-*cmpCD* construct maps, shown in Figure 1. The specific constructs on the plasmid maps are explained in further detail in the following sections.

![Figure 1](https://static.igem.wiki/teams/5624/f/experiment-fig-1.webp)

Figure 1. Plasmid maps of the ppsbA1-*cmpAB* and ppsbA1-*cmpCD* constructs

#### *cmpAB* and *cmpCD* Fusion Genes

In the native system, the *cmpABCD* operon is transcribed and translated into four individual proteins—*cmpA*, *cmpB*, *cmpC*, and *cmpD*—that then assemble to form the BCT1 complex responsible for ATP-driven HCO3- uptake. For this project, the team engineered two fusion genes, *cmpAB* and *cmpCD*, rather than expressing the *cmpA*, B, C, and D compartments separately.

Based on the functional analysis conducted by Rottet et al. on multiple BCT1 mutants in E. Coli, where the HCO3- uptake rates for a subset of seven genetic constructs were measured, the *cmpCD* fusion gene exhibited the highest uptake rate. Our team thereby hypothesized that by designing the *cmpABCD* operon into two fusion genes—*cmpAB* and *CD*, the speed of BCT1 transporter complex formation (as *cmpAB* and *CD* fusion proteins) could be enhanced, where the resulting polypeptides may promote more rapid and stable assembly of BCT1 and thereby facilitate the rate of HCO3- docking onto *cmpA*. In addition to an increased complex formation speed, the *cmpAB* fusion protein dimers (*cmpAB* + *cmpAB* combination after protein expression) would contain multiple substrate-binding proteins (SBPs), where one *cmpAB* dimer would consist of two SBPs, further contributing to increasing the rate of HCO3- transport and ultimately justifying the feasibility of our fusion gene designs.

#### ppsbA1 Promoter

The team used ppsbA1, a strong light-response promoter, to drive the downstream of the fusion genes, *cmpAB* and *cmpCD*, respectively. The native ppsbA1 promoter drives the expression of one of three psbA genes that encode the D1 protein, an essential component of Photosystem II (PSII), in both *Synechococcus elongatus* PCC 6803 and PCC 7942. PSII plays a crucial role in photosynthesis by catalyzing the splitting of water molecules into oxygen and protons, a vital step in converting light energy into chemical energy (Sacharz et al., 2009).

The ppsbA1 promoter behaves differently in *S. elongatus*PCC6803 and PCC7942. In the strain PCC6803, the ppsbA1 is manipulated by both light and CO2 levels. However, in PCC7942, the ppsbA1 promoter is strongly activated at normal (low to moderate) light, but demonstrates reduced activity under strong light intensities (Van der Staay et al., 1991; Nair et al., 2001). Given this inherent feature of the ppsbA1 promoter, the team can thereby easily manipulate the light intensity levels to enhance ppsbA1-*cmpAB* and *CD* fusion gene activity more efficiently.

#### Kanamycin: An Antibiotic-Resistant Gene

Kanamycin, a commonly used selective agent for bacteria, is an antibiotic that binds to the 30S subunit of bacterial ribosomes to disrupt protein synthesis. This results in the production of faulty proteins, ultimately leading to the death of bacterial cells. For our project, the ppsbA1 promoter construct contains the kanamycin-resistant gene, which enables the competent bacteria to survive on LB-Kan plates, allowing for the selection of cells containing the desired gene (Chulluncuy et al., 2016).

#### His-tag: Protein Detection

To detect and validate gene expression and activity, both the *cmpAB* and *CD* fusion genes were also cloned with a polyhistidine tag (His-tag for short) for protein detection. A His-tag is a short tract of histidine residues genetically fused to the N-terminus or C-terminus of a target protein, which provides a means of detecting and purifying the recombinant protein (Kielkopf et al., 2020). For our project, we cloned the *cmpAB* and *CD* fusion genes with a 6x N-terminal His-tag. With the use of anti-His antibodies, the size and presence of our His-tagged BCT1 protein assembly could be detected via Western blotting. Therefore, the addition of a His-tag serves as a universal detection system that confirms successful *cmpABCD* gene expression driven by the light-inducible promoter ppsbA1. Additionally, His-tag usage eliminates the need to generate separate antibodies against the *cmpAB* and *CD* fusion proteins, serving as an efficient approach to confirm our vector constructs.

A cloning overview of the ppsbA1-*cmpAB* and *CD* fusion genes is shown in Figure 2.

![Figure 2](https://static.igem.wiki/teams/5624/f/experiment-fig-2.webp)

Figure 2. A schematic diagram of the team’s ppsbA1-*cmpAB* and ppsbA1-*cmpCD* fusion genes’ cloning procedure.

### Confirmation of our Genetically Engineered Composition Parts

#### [Fusion Genes PCR](https://static.igem.wiki/teams/5624/f/2x-pcr-mix-protocol.pdf)

The team requested the Mission Biotechnology company in Taiwan to synthesize our *cmpAB* and *cmpCD* fusion genes, which were then amplified via PCR. For the two fusion genes, the team designed the forward primer flanked with the AvrII restriction site and the reverse primer flanked with the BamHI restriction site to facilitate cloning downstream of the ppsbA1 promoter. We further conducted 1% [gel electrophoresis](https://static.igem.wiki/teams/5624/f/2x-pcr-mix-protocol.pdf) to verify the PCR and gene expression success.

#### [Double-enzyme Digestion](https://static.igem.wiki/teams/5624/f/neb-enzyme-digestion.pdf)

After the *cmpAB* and *cmpCD* fusion genes were amplified via PCR, the AvrII and BamHI double enzymes were simultaneously used to digest the *cmpAB* and *cmpCD* PCR products along with the ppsbA1 promoter plasmid, incubated at 37°C for 1 hour. The enzymes were then inactivated via incubation at 65°C for 20 minutes.

#### [T4 Ligation](https://static.igem.wiki/teams/5624/f/t4-dna-ligation-protocol.pdf)

The T4 DNA ligase was used to join the digested *cmpAB* or *cmpCD* fusion gene PCR product with the digested ppsbA1 promoter plasmid, creating the ppsbA1-*cmpAB* and ppsbA1-*cmpCD* plasmids.

#### [Bacterial Transformation](https://static.igem.wiki/teams/5624/f/bacterial-transformation.pdf)

The ligated ppsbA1-*cmpAB* and ppsbA1-*cmpCD* plasmids were transformed into DH5α competent bacterial cells after [bacterial plasmid extraction](https://static.igem.wiki/teams/5624/f/bacteria-plasmid-extraction.pdf) was performed, separately, via 42°C heat shock for 45 seconds, followed by plating on LB agar plates with 50 μg/mL kanamycin for selection. The bacterial colonies observed on the LB-Kan plates indicated that they contained the team’s synthesized plasmids, which were subsequently isolated for verification.

#### Verifying PCR Products

The bacterial colonies observed on the LB-Kan plates were inoculated to perform PCR via the same forward/reverse primers of *cmpAB* and *cmpCD* that the team designed. Then, we conducted 1% gel electrophoresis for our PCR products to check whether the bacteria contained the construct the team created.

### Cyanobacterial Transformation

Cyanobacterial transformation was performed to allow *S. elongatus* PCC 7942 containing either the psbA1-*cmpAB* or ppsbA1-*cmpCD* plasmids. Several methods were attempted to transform our plasmids into cyanobacteria cells in the form of spun-down liquid culture concentrates.

The team first performed cyanobacterial chemical transformation. Inspired by the iGEM team at Hong Kong University and Riaz et. al, Our chemical transformation involved the use of CaCl2 and heat shock to generate pores in the cell membrane, theoretically allowing the plasmid to bypass the membrane, enter the cell, and express the genes it carries (Riaz et. al, 2022). Second, we also did natural transformation, which uses our strain’s natural ability of taking in plasmids as a substitute for heat shock. It consists of an intake day and a recovery day, where plasmids are first added and then BG-11 (Riaz et. al, 2022). Lastly, another way of introducing our plasmids into the cell is via electroporation, which uses short high-voltage pulses to transiently permeabilize cell membranes and allow DNA uptake. Following recovery in BG-11 medium, cells were plated on selective agar, where transformants typically appeared within 1–2 weeks (Matsuoka et. al, 2001).

### Wild-type Cyanobacteria: Functional Analyses

The data retrieved from the functional analyses of wild-type cyanobacteria are integrated into our model in MATLAB, which enables us to model the inorganic carbon uptake efficiency of the cyanobacteria.

#### [CO2 Assay](https://static.igem.wiki/teams/5624/protocol/co2-assay.pdf)

The CO2 assay was designed to assess the ability of *S. elongatus* PCC 7942 to reduce environmental CO2 concentrations over time. Cyanobacteria samples were divided randomly into engineered/wild-type/ BG-11 only (control) groups. Cultures were ensured to have an OD730 value of at least 0.3 prior to experiment then sealed with a gaseous CO2 sensor to monitor headspace CO2 levels for one hour post-exposure.

#### [HCO3-Assay](https://static.igem.wiki/teams/5624/protocol/hco3-assay.pdf)

To directly measure the bicarbonate uptake capacity of *S. elongatus* PCC 7942, we designed a BaCl2 precipitation assay. This method is based on the principle that Ba2+ ions only react with CO32- ions among all inorganic carbons, which allows precise quantification of HCO3-. Standard curves were first generated using known NaHCO3 concentrations to correlate absorbance values with the formation of BaCO3 based on the Beer-Lambert law. Cyanobacteria samples were divided randomly into engineered/wild-type/ BG-11 only (control) groups. The experimental samples from cultures incubated with 30µM NaHCO3 were put under FL3OSSEX-D-X QUAD PHOSPHOR T8 light. Every 30 minutes, samples were collected and syringe-filtered, then mixed with 1M BaCl2. The mixture was left to stand until the turbidity reached a stable value, indicating that all available inorganic carbon had fully precipitated as BaCO3 when the absorbance no longer increased at this point. The resulting turbidity of the mixture was measured at 997.5 nm using a Nabi UV/Vis Nano Spectrophotometer every 30 seconds until the data stabilized, and the final stable value was used. Lastly, the residual extracellular HCO3- concentrations were determined by plotting the absorbance value against the standard curve.

#### [O2 Assay](https://static.igem.wiki/teams/5624/protocol/o2-assay.pdf)

Because oxygen evolution was a direct proxy for photosynthetic activity, we performed an assay to quantify the O2 release under light-induced conditions. Cultures were illuminated with white light, and O2 production was measured over 120 minutes. Data points were collected every 10 minutes. Rates were then normalized to OD750 and chlorophyll a content, determined spectrophotometrically using methanol extraction and absorbance at 665 and 720 nm.

#### [pH Assay](https://static.igem.wiki/teams/5624/protocol/ph-assay.pdf)

Because pH is closely linked to inorganic carbon dynamics and the survival of cyanobacteria, we performed a pH assay to monitor pH changes in the culture medium for CO2 dissolution and bicarbonate utilization. Cyanobacteria cultures or BG-11 (control) were exposed to ambient air CO2 levels under illumination. A stick pH meter was inserted directly into the liquid cultures, and pH was measured every 10 minutes over a 2-hour period. Shifts in pH reflected the balance between CO2 uptake, HCO3- assimilation, and metabolic byproducts, providing an indirect but complementary readout of carbon capture efficiency in live cultures.

### Expression Confirmation

#### Overview

After constructing the two plasmids, ppsbA1-*cmpAB* and ppsbA1-*cmpCD*, the team designed several tests to verify their activities in our cyanobacterial strain—*Synechococcus elongatus* PCC 7942. The tests included  [RT-qPCR](https://static.igem.wiki/teams/5624/f/rt-qpcr-protocol.pdf)  to detect mRNA expression levels of the *cmpAB* and *cmpCD* fusion genes, respectively, and a planned western blot analysis to assess the BCT1 fusion protein expression level in the engineered *S. elongatus*PCC 7942 after successful transformation of the *cmpAB* and *CD* fusion compartments by probing the protein with an anti-His antibody.

Having confirmed that the two plasmids containing the fusion genes *cmpAB* and *cmpCD* were expressed adequately under the control of the light-dependent ppsbA1 promoter in *S. elongatus* PCC 7942, the team planned to proceed and analyze whether the *cmpAB* and *cmpCD* fusion proteins enhances carbon fixation rates by performing RT-qPCR to detect the mRNA level of RuBisCo, a key enzyme of the Calvin cycle integral in determining the speed of cyanobacterial photosynthesis. An increase in the mRNA levels of RuBisCo further indicates an increased carbon fixation rate.

#### Time Course Sample Collection

To monitor the CO2 fixation efficiency of *S. elongatus* PCC 7942, we used a CO2 sensor to detect the CO2 concentration of sealed culture flasks. Cyanobacteria samples were divided randomly into engineered/wild-type/BG-11 only (control) groups. The team conducted a light induction time course to evaluate cyanobacteria’s responsiveness to white light compared to dark conditions. Samples were cultured in BG-11 medium and collected at 0 hours, 0.5 hours, 1 hour, 1.5 hours, and 2 hours post-induction. These samples were then used for further analyses, including RT-qPCR and western blotting.

![Figure 3](https://static.igem.wiki/teams/5624/f/experiment-fig-3.webp)

Figure 3. A schematic diagram of the time course sample collection and functional assays the team wanted to perform

#### RT-qPCR for *cmpAB* & *cmpCD* fusion genes and RuBisCO

Experimental samples of cyanobacteria containing either the ppsbA1-*cmpAB* or ppsbA1-*cmpCD* constructs, as well as control groups consisting of cyanobacteria alone or cyanobacteria carrying only the ppsbA1 promoter plasmid, were cultured in BG-11 medium for the collection of time-course samples, as described above. The levels of *cmpAB* and *cmpCD* mRNA expression were evaluated by extracting RNA from these samples and performing RT-qPCR analysis. The team designed qPCR primers that specifically targeted the junction regions of the fusion genes, *cmpAB* and *cmpCD*. mRNA induction levels were normalized using the endogenous control gene SecA to serve as an internal reference, ensuring consistent sample loading and accurate comparison across conditions. To confirm the team’s constructs would be induced, driven by a light-dependent promoter, ppsbA1, the team compared mRNA induction levels of *cmpAB* and *cmpCD* at 0.5 hrs, 1 hour, 1.5 hours, and 2 hours to 0 hr in the experimental samples, respectively. To further verify whether the mRNA induction levels of *cmpAB* and *cmpCD* enhance the mRNA induction level of RuBisCO for the acceleration of carbon fixation, the team compared the mRNA induction of RuBisCO at different time courses in the experimental samples to the mRNA level of secA, an internal control. The team expected to detect an increase in the mRNA level of RuBisCO in the experimental samples exposed to light compared to the controls.

#### Western blot analysis for the *cmpAB* and *cmpCD* fusion proteins

The team collected time-course samples of cyanobacteria containing the experimental constructs *psbA*1-*cmpAB* and *psbA*1-*cmpCD*, along with controls consisting of wild-type cyanobacteria and cyanobacteria carrying the ppsbA1 promoter alone. Expression of the His-tagged *cmpAB* and *cmpCD* fusion proteins was confirmed by western blot analysis, as both constructs included a 6-His tag immediately downstream of the start codon. Total proteins were extracted from induced cultures at various time courses using the cell lysis buffer. Protein concentrations were measured using the Bradford assay. Equal amounts of protein were loaded onto 10% SDS-PAGE gels and separated by electrophoresis, after which the proteins were transferred onto PVDF membranes. Following blocking, membranes were probed with anti-His-tag primary antibodies, then incubated with HRP-conjugated anti-rabbit secondary antibodies. Protein bands at the expected molecular weights were visualized by chemiluminescence. The actin antibody was used as an internal control to ensure equal protein loading and to normalize target protein levels during western blot analysis.

### Additional Experiments

#### Polyvinyl Alcohol Hydrogel (PVA): An Organismal Adherence Platform

Additional to genetically engineering cyanobacteria and developing a mathematical model that computes and explains its increased efficacy in carbon fixation, our team initially envisions a carbon fixation chamber containing cyanobacteria adhered to polyvinyl alcohol (PVA) hydrogel in industrial applications. On one hand, the transparency, porosity, and biocompatibility of the PVA hydrogel optimize light and inorganic carbon exposure. On the other hand, the chamber design offers a supportive environment that provides cyanobacteria with nutrient supply and gas exchange. Ultimately, we expected the system to offer a scalable and sustainable solution for mitigating atmospheric CO2 pollution by converting it into biomass through enhanced photosynthesis. For the team’s currently optimized PVA hydrogel design, a 6 wt% PVA solution was prepared by gradually adding PVA powder (MW 85,000-145,000; 99% hydrolyzed) into 94 mL of 95 °C deionized water, which was stirred until fully dissolved. The solution was cooled to room temperature, and its pH was adjusted to 6.5 using HCl and a pH meter. The chemical crosslinker, glutaraldehyde (GA, 0.1 mol GA per mol PVA –OH), was then added. The resulting PVA–GA mixture was poured into a rectangular plastic mold and incubated overnight to promote gelation at 37 °C.” Then add another paragraph: “During our PVA hydrogel optimization process and characteristic analysis attempts, we actively reached out to numerous specialists in materials science. Dr. Hintze, a researcher at the University of Bonn, provided us with insights into feasible qualification methods, such as flow resistance and bacterial migration assays, when we were fixated on measuring nanoscopic pore sizes. Dr. Matsumura, a professor from the Japan Advanced Institute of Science and Technology, specializes in functional polymeric biomaterials and biomedical engineering. Through our online meeting with him and inquiries about his past experimental study on comparing conventional DMSO-based PVA hydrogels and novel hot-pressed ones (Sakaguchi et al., 2017). From Dr. Matsumura, we learned that hydrogel transparency is dependent on pore size, which arises from physical crosslinking and crystallizing processes. He also advised us on DMSO detoxification methods, including freeze-drying and ethanol immersion. Regarding quantitative analyses, Dr. Matsumura turned us to tensile testing (measuring Young’s modulus) and assessing cell viability through spectrophotometry.

### Protocols

[Bacterial Transformation](https://static.igem.wiki/teams/5624/f/bacterial-transformation.pdf)

[Bacterial Plasmid Extraction](https://static.igem.wiki/teams/5624/f/bacteria-plasmid-extraction.pdf)

[Gel Electrophoresis](https://static.igem.wiki/teams/5624/f/gel-electrophoresis-2025.pdf)

[RT-qPCR](https://static.igem.wiki/teams/5624/f/rt-qpcr-protocol.pdf)

[NEB Enzyme Digestion](https://static.igem.wiki/teams/5624/f/neb-enzyme-digestion.pdf)

[T4 Ligation](https://static.igem.wiki/teams/5624/f/t4-dna-ligation-protocol.pdf)

[2X-PCR Mix](https://static.igem.wiki/teams/5624/f/2x-pcr-mix-protocol.pdf)

[Polyvinyl Hydrogel(PVA)](https://static.igem.wiki/teams/5624/f/polyvinyl-hydrogel-pva.pdf)

[CO2 Assay](https://static.igem.wiki/teams/5624/protocol/co2-assay.pdf)

[HCO3- Assay](https://static.igem.wiki/teams/5624/protocol/hco3-assay.pdf)

[O2 Assay](https://static.igem.wiki/teams/5624/protocol/o2-assay.pdf)

[pH Assay](https://static.igem.wiki/teams/5624/protocol/ph-assay.pdf)

## References

Chen, Y., Li, J., Lu, J., Ding, M., & Chen, Y. (2022). Synthesis and properties of Poly (vinyl alcohol) hydrogels with high strength and toughness. Polymer Testing, 108, 107516.

Chulluncuy, R., Espiche, C., Nakamoto, J. A., Fabbretti, A., & Milón, P. (2016). Conformational response of 30S-bound IF3 to A-site binders streptomycin and kanamycin. Antibiotics, 5(4), 38.

Kielkopf, C. L., Bauer, W., & Urbatsch, I. L. (2020). Purification of polyhistidine-tagged proteins by immobilized metal affinity chromatography. Cold Spring Harbor Protocols, 2020(6), pdb-prot102194.

Koropatkin, N., Koppenaal, D., Pakrasi, H., & Smith, T. (2007, January). The Structure of a Cyanobacterial Bicarbonate Transport Protein, *cmpA*\*—Journal of Biological Chemistry. https://www.jbc.org/article/S0021-9258(20)72131-9/fulltext

Kupriyanova, E. V., Pronina, N. A., & Los, D. A. (2023). Adapting from Low to High: An Update to CO2-Concentrating Mechanisms of Cyanobacteria and Microalgae. Plants, 12(7), 1569. https://doi.org/10.3390/plants12071569

Li, J., Ai, H., Wang, Q., He, H., Chang, X., Chen, G., ... & Qin, F. (2025). Optimization of the Preparation Process of Crosslinked Polyvinyl Alcohol and Its Thermal Stability in Cementing Slurry. Gels, 11(2), 98.

Sakaguchi, T., Nagano, S., Hara, M., Hyon, S. H., Patel, M., & Matsumura, K. (2017). Facile preparation of transparent poly (vinyl alcohol) hydrogels with uniform microcrystalline structure by hot-pressing without using organic solvents. Polymer journal, 49(7), 535-542.

Matsuoka, M., Takahama, K., & Ogawa, T. (2001). Gene replacement in cyanobacteria mediated by a dominant streptomycin-sensitive rps12 gene that allows selection of mutants free from drug resistance markers. Microbiology, 147(8), 2077-2087.

Omata, T., Price, G. D., Badger, M. R., Okamura, M., Gohta, S., & Ogawa, T. (1999). Identification of an ATP-binding cassette transporter involved in bicarbonate uptake in the cyanobacterium Synechococcus sp. Strain PCC 7942. Proceedings of the National Academy of Sciences of the United States of America, 96(23), 13571–13576. https://doi.org/10.1073/pnas.96.23.13571

Price, G. D., Badger, M. R., Woodger, F. J., & Long, B. M. (2008). Advances in understanding the cyanobacterial CO2-concentrating-mechanism (CCM): functional components, Ci transporters, diversity, genetic regulation, and prospects for engineering into plants. Journal of Experimental Botany, 59(7), 1441–1461. https://doi.org/10.1093/jxb/erm112

Riaz, S., Jiang, Y., Xiao, M., You, D., Klepacz-Smółka, A., Rasul, F., & Daroch, M. (2022). Generation of miniploid cells and improved natural transformation procedure for a model cyanobacterium *Synechococcus elongatus* PCC 7942. Frontiers in microbiology, 13, 959043. https://doi.org/10.3389/fmicb.2022.959043

Rottet, S., Rourke, L. M., Pabuayon, I., Phua, S. Y., Yee, S., Weerasooriya, H. N., ... & Price, G. D. (2024). Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts. Journal of Experimental Botany, 75(16), 4926-4943.

Wang, M., Bai, J., Shao, K., Tang, W., Zhao, X., Lin, D., ... & Ye, J. (2021). Poly (vinyl alcohol) hydrogels: the old and new functional materials. International Journal of Polymer Science, 2021(1), 2225426.

Zhang, Z., Liu, Y., Lin, S., & Wang, Q. (2020). Preparation and properties of glutaraldehyde crosslinked poly (vinyl alcohol) membrane with gradient structure. Journal of Polymer Research, 27(8), 228.

Show all

* [PCR Confirmation](#PCR)
* [Light-Induced Expression](#Light-Induced-Expression)
* [Assay Tests](#assay-tests)
* [PVA Hydrogel Optimization](#PVA-optimization)
* [Conclusion](#conclusion)
* [Future Directions](#future-directions)
* [References](#references)

### 1. PCR Confirms Successful Amplification of *cmpAB* and *cmpCD* fusion genes synthesized from the Mission Biotech Company

#### 1.1 Confirming the *cmpAB* and *cmpCD* PCR Products

The team designed two sets of forward and reverse primers accompanying the *cmpAB* (BBa\_25SKOH98) and *cmpCD* (BBa\_25N38C54) fusion gene sequences, which served as basic genetic parts. The AvrII enzyme cut site was introduced on the 5’ end of the forward primers, and the BamHI enzyme cut site was introduced on the 3’ end of the reverse primers, together allowing fusion gene generation via PCR. After conducting PCR on the separate *cmpAB* and *cmpCD* constructs, purchased from the Mission Biotech company, the team performed gel electrophoresis to verify the PCR products on a 1% agarose gel. As shown in Figure 1, Lane 1 was a 1kb DNA ladder as a reference. Lanes 2 and 4 were the original plasmids that contained the two fusion genes, which were *cmpAB* and *cmpCD*, respectively. PCR products showed single distinct DNA bands after PCR amplification, where *cmpAB* had a length of 2202 base pairs (bp) in lane 3 and *cmpCD* had a length of 2823 bp in lane 5. To summarize, the team obtained successful results, indicating that the PCR products of the *cmpAB* and *cmpCD* fusion genes were generated.

![Figure 1](https://static.igem.wiki/teams/5624/result/result-fig-1.webp)

Figure 1: Gel image as a confirmation of the successful PCR products of the basic parts, *cmpAB* (BBa\_25SKOH98) and *cmpCD* (BBa\_25N38C54). Lane 1 was a 1kb DNA ladder as a reference, which the DNA fragments correspond to. Lanes 2 and 4 were the original plasmids that contained the two fusion genes (*cmpAB* and *cmpCD*) from the Mission Biotech Company. Lane 3 was the *cmpAB* PCR product, 2202bp. Lane 4 showed the *cmpCD* PCR product, 2823bp.

#### 1.2 Confirmation of the Composite Parts—the ppsbA1-*cmpAB* Construct (BBa\_25K6M3E8) and ppsbA1-*cmpCD* Construct (BBa\_25ZLGSPR) after Bacterial Transformation

##### 1.2.1 Overview

Following bacterial (E. Coli DH5ɑ) transformation, individual bacterial colonies were selected to perform PCR and then subjected to full-length *cmpAB* and *cmpCD* PCR amplification, as shown in Figures 2 and 3.

##### 1.2.2 ppsbA1-*cmpAB* Construct (BBa\_25K6M3E8)

The team performed bacterial transformation on LB-Kanamycin selection plates and inoculated several bacterial colonies that were later subjected to PCR analysis using the same primer set as shown in Figure 2, allowing us to amplify the *cmpAB* fusion gene (Fig. 6). In lane 8, the team used the original construct containing *cmpAB*, obtained from the Mission Biotech company, as a control, showing a 2.2 kb distinct band. The bacterial colonies' PCR products, ran on 1% gel electrophoresis, showed that bacterial colonies #1, 2, 3, 4, and 6, which correspond to lanes 2, 3, 4, 5, and 7, each produced a distinct single 2.2 kb band, indicating that the bacterial colonies contained the team’s pspbA1-*cmpAB* composite part. (BBa\_25K6M3E8). To conclude, the results confirmed that the composite part ppsbA1-*cmpA*B(BBa\_25K6M3E8) was successfully constructed.

![Figure 2](https://static.igem.wiki/teams/5624/result/result-fig-2.webp)

Figure 2: Gel image confirming that the composite part ppsbA1-*cmpAB* (BBa\_25K6M3E8) was successfully constructed. Lane 1 was a 1kb DNA ladder as a reference, which the DNA fragments correspond to. Lane 8 was the original construct containing *cmpAB*, obtained from the Mission Biotech company. The construct was used as a positive control for PCR analysis. Lanes 2, 3, 4, 5, and 7, which correspond to bacterial colonies # 1, 2, 3, 4, and 6, showed a distinct single 2.2kb band after PCR. Lane 6 was the bacterial colony #5. With an absence of a distinct band as proof for PCR products, the lane confirmed that colony #5 did not take up the team’s ppsbA1-*cmpAB* composite part.

##### 1.2.3 ppsbA1-*cmpCD* construct (BBa\_25ZLGSPR)

The team performed bacterial transformation on LB-Kanamycin selection plates and inoculated several bacterial colonies to perform PCR analysis using the same primer as in Figure 2. In Figure 3, lane 1 was a 1 kb DNA ladder as a reference. Lane 6 was the original construct containing *cmpCD* as a positive control, obtained from the Mission Biotech company, which showed a band between 2.5 kb and 3 kb after PCR analysis. Lanes 3, 4, and 5 corresponded to the bacterial colonies # 2, 3, and 4, which showed a distinct single band that had the same size (2.8 kb) as the control in lane 6 after PCR analysis, indicating that these bacterial colonies contained the team’s full-length *cmpCD* fusion gene composite part, ppsbA1-*cmpCD* (BBa\_25ZLGSPR). Lane 2 contained the bacterial colony #1, which produced a PCR product greater than 10 kb after PCR. This indicated that bacterial colony #1 did not contain the team’s composite part ppsbA1-*cmpCD* (BBa\_25ZLGSPR) since its PCR product was way larger than the *cmpCD* fusion gene side, 2.8 kb. To conclude, the figure results confirmed that the composite part ppsbA1-*cmpCD* (BBa\_25ZLGSPR) was successfully constructed.

![Figure 3](https://static.igem.wiki/teams/5624/result/result-fig-3.webp)

**Figure 3. Gel image confirming that the composite part ppsbA1-*cmpCD* (BBa\_25ZLGSPR) was successfully constructed.** Lane 1 was a 1 kb DNA ladder as a reference, which the DNA fragments correspond to. Lane 6 was the original construct containing *cmpCD* used as a positive control for PCR analysis. Lanes 3,4, and 5, which corresponded to bacterial colonies # 2, 3, and 4, showed a distinct single band between 2.5 kb and 3 kb (using the DNA ladder as a reference) after PCR. The bands generally corresponded to *cmpCD*’s 2.8kb band, indicating bacterial colonies #2, 3, and 4 contained the team’s composite part, ppsbA1-*cmpCD* (BBa\_25ZLGSPR). Lane 2 corresponded to bacterial colony #1, producing a PCR product above 10 kb that deviated from the 2.8 kb in the lane 6 control group, indicating that this bacterial colony did not contain the team’s composite part ppsbA1-*cmpCD* (BBa\_25ZLGSPR).

### 2. RT-qPCR Analysis Shows No Endogenous Light-Induced Expression of *cmp* Genes in Wild-Type Cyanobacteria, *S. elongatus* PCC 7942

This RT-qPCR experiment serves as a negative control to confirm the baseline expression of target genes in the wild-type cyanobacteria strain *S. elongatus PCC 7942*. Because the wild-type strain does not contain the team’s engineered constructs (BBa\_25K6M3E8 (ppsbA1-*cmpAB*) or BBa\_25ZLGSPR (ppsbA1-*cmpCD*)) the ppsbA1 promoter is absent, and therefore no light-induced mRNA expression of *cmpA*, *cmpB*, *cmpC*, or *cmpD* was expected. In other words, the experiment aimed to verify that light exposure alone would not trigger transcription of these genes in unmodified cyanobacteria cells.

The wild-type PCC 7942 cells were cultured in BG-11 medium until the optical density at 730 nm (OD730) reached 0.4 or 0.5. Time-course samples were collected at 0 min, 30 min, 60min, 90 min, 120 min in the presence of light. The housekeeping gene *secA* was used as an internal control to normalize mRNA levels across the different time points. To detect mRNA induction of *cmpA*, *cmpB*, *cmpC*, *cmpD*, and RuBisCO genes, specific primer pairs were designed for each target gene, and a common primer set targeting *secA* was used to quantify *secA* mRNA as an internal control.

To analyze RT-qPCR data, different genes,*cmpA*, *cmpB*, *cmpC*, *cmD*, and RuBisCO, were separated along the X-axis, and distinct colors represent various time courses as shown in Figure 4. The red bar indicated control samples kept in the dark; the sky blue bar represented samples exposed to light for 30 minutes; the gray bar represented samples under light exposure for 60 minutes; the green bar represented samples under light exposure for 90 minutes, and finally, the orange bar represented samples with 120 minutes of light exposure.

The team measured mRNA induction levels of *cmpA*, *cmpB*, *cmpC*, *cmD*, and RuBisCO across all time course samples and compared them to the control samples kept in the dark. As shown in Figure 4, no significant differences in the mRNA induction were observed for these genes at 30 mins, 60 mins, 90 mins, and 120 mins of light exposure. Overall, the data aligned with the team’s expectations that no gene exhibited significant mRNA induction across the various light exposure time course samples, and the outcome was consistent with the fact that wild-type PCC7942 did not obtain the composite parts created by the team.

![Figure 4](https://static.igem.wiki/teams/5624/result/result-fig-4.webp)

**Figure 4:** The mRNA induction levels of *cmpA*, *cmpB*, *cmpC*, *cmD*, and RuBisCO were detected at 30 mins, 60mins, 90 mins, and 120 mins of light exposure and compared to controls maintained in the dark. No significant differences in the mRNA levels of these genes were observed between the light-exposed samples and the controls.

### 3. CO2 Assay Presents a Constant Carbon Fixation Rate in *Synechococcus elongatus* PCC 7942

![Figure 5](https://static.igem.wiki/teams/5624/result/result-fig-5.webp)

**Figure 5: CO2 concentration change of wild-type cyanobacteria and BG-11 medium over time.** The “With Cyanobacteria” group contained a 100 mL liquid culture of *S. elongatus* PCC 7942 that reached an OD730 of 0.5. The “BG-11 Only” group was a 250 mL flask that contained only 100 mL of BG-11 medium. ​​The horizontal and oblique asymptotes are marked in thin black lines.

#### 3.1 Overview

In this experiment, both groups had a decreasing CO2 concentration over time, the same results as we predicted. Specifically, the “With Cyanobacteria” group had a lower final CO2 concentration of 316 ppm, compared to 398 ppm in the “BG-11 Only” group. Samples are taken once per two seconds.

#### 3.2 Discussion

In figure 5, the team observed that the “BG-11 Only” group, as a control, approached a horizontal asymptote, indicating the establishment of a CO2 equilibrium between the BG-11 medium and the air inside the flask. On the other hand, the “With Cyanobacteria” group presented a different trend. First, the “With Cyanobacteria” group exhibited a near-exponential decrease in CO2 concentration, which probably resulted from the combined effects of both equilibration and carbon fixation. Then, the graph approached an oblique asymptote, meaning that the carbon fixation rate is constant after equilibrium is reached.

### 4. HCO3- Precipitation Assay Shows that No Porins Exist in the Outer Membrane of the Cyanobacteria, *S. elongatus* PCC 7942

#### 4.1 Overview

To evaluate the bicarbonate uptake ability of *S. elongatus* PCC 7942, the team implemented a BaCl2 precipitation assay, as Ba2+ ions only bind with CO32- ions but not others. The team first created linear calibration curves of Absorbance/[BaCO3] for 10 mM, 15 mM, and 25 mM concentrations by measuring BaCO3 formation from known NaHCO3 concentrations based on the Beer-Lambert law. We then used peak absorbance numbers at OD998 to detect the concentration of NaHCO3 change in cyanobacteria, S. elongatus PCC 7942, for different incubation times. As shown in Figure 6, the concentration of extracellular NaHCO3 does not vary for different incubation times. The regression analysis yielded R2=0.007, indicating no measurable correlation between bicarbonate concentration and incubation time.

![Figure 6](https://static.igem.wiki/teams/5624/result/result-fig-6.webp)

**Figure 6: Change in Concentration of NaHCO3 from OD998 over different incubation times.** The concentration of extracellular NaHCO3 was measured every half an hour in a time course experiment. The regression analysis yielded R2=0.007, indicating no measurable correlation between bicarbonate concentration and incubation time.

#### 4.2 Discussion

This result demonstrated that extracellular HCO3- concentration remained constant all the time during lighting, suggesting that wild-type cyanobacteria did not take up HCO3- from the surrounding area. Therefore, the team concluded that inorganic carbon uptake in the team’s strain does not occur via HCO3- transport through outer membrane porins but instead relies solely on simple diffusion of CO2, which was consistent with the team’s assumptions from our modeling analyses.

### 5. pH Assay Demonstrates Stable Medium Conditions During Carbon Fixation in Cyanobacteria, *S. elongatus* PCC 7942, cultured cells

#### 5.1 Overview

To show whether there are changes in pH in the cyanobacteria culture grown in BG-11 medium, the team detected pH by a pH meter every 10 minutes up to 120 minutes under ambient temperature and room CO2 conditions (400 ppm). As shown in Figure 7, the pH remained relatively stable, with only minor fluctuations within a narrow and acceptable range of pH between 7.3-7.6.

![Figure 7](https://static.igem.wiki/teams/5624/result/result-fig-7.webp)

**Figure 7: Time in minutes during incubation vs. the change of pH in the medium over time.** The data showed the pH remained relatively stable at a range of pH between 7.3-7.6.

#### 5.2 Discussion

Overall, the data indicates the culture medium maintained a stable pH throughout the incubation time and the carbon-fixing related chemical reactions. This stability is critical for future usages of the team’s cyanobacteria in the BG-11 medium form, as pH directly affects the efficiency and survival of cyanobacteria (Ito-Miwa, 2025).

### 6. O2 Assay Confirms Normal Photosynthetic Activity in Cyanobacteria, *S. elongatus* PCC 7942, cultured cells

#### 6.1 Results

The team also investigated O2 concentrations in the BG-11 medium cultured cyanobacteria, S. elongatus PCC 7942, to confirm photosynthesis activity. In Figure 8, the data showed that the O2 concentration gradually increased in cyanobacteria under continuous white light illumination for over 80 minutes. The steady linear increase in dissolved O2 concentration indicated active photosynthetic oxygen production. The regression analysis also yielded a strong positive linear correlation, suggesting an increasing O2 concentration.

![Figure 8](https://static.igem.wiki/teams/5624/result/result-fig-8.webp)

**Figure 8: Change in O2 concentration in mg/L over time.** R2=0.9196. The graph was normalized with chlorophyll concentration. The data showed the steady O2 concentration increasing, indicating that the cyanobacterial cultures had photosynthetic activity.

#### 6.2 Discussion

These results confirmed that the cyanobacterial cultures were metabolically active and photosynthetically competent, demonstrating that light intensity and culturing conditions were sufficient to sustain normal oxygenic photosynthesis, an important aspect of cyanobacteria’s survival.

### 7. Additional Experimental Results: PVA Hydrogel Optimization Identifies Conditions for Ideal Hydrogel Formation as an Organismal Adherence Platform

#### 7.1 Overview

Initially, the team was planning and devising a polyvinyl alcohol (PVA) hydrogel adherence platform for cyanobacterial immobilization in the team’s carbon fixation chamber device. The team focused on optimizing five hydrogel characteristics: transparency, porosity, tensile strength, water retention, and biocompatibility, which help ensure proper, robust growth of the engineered cyanobacteria with ideal light transmittance, nutrient levels, mechanical support, and viability. The team experimented with various factors of influence, broadly categorized into organic cosolvent combinations, PVA concentrations, and additive concentrations (as shown in the following tables), to modify and enhance the characteristics mentioned above (Chen et al., 2022).

#### 7.2 Organic Cosolvent Addition

When dissolving PVA powder, using a mixed solvent of water and water-miscible organic solvents could enhance the tensile strength, water retention, and transparency of PVA hydrogels (Hassan & Peppas, 2018). The addition of organic cosolvents is accompanied by physical crosslinking, a process that involves facilitating crystallite formations (physical crosslinking points) within the PVA hydrogel structure, allowing the mechanical load of the hydrogels to be evenly distributed along the crystallites (Hassan & Peppas, 2018). In the team’s experiments, the freeze-thaw (FT) method was employed for physical crosslinking, where cycles of freezing and thawing of the PVA solution promote crystallization. As the number of cycles increased, the amount and stability of crystallites should also increase.

Some organic cosolvents conventionally used in PVA hydrogel production for functionality modification purposes included dimethyl sulfoxide (DMSO), ethylene glycol (a diol), and glycerol (a triol). Specifically, the addition of ethylene glycol, a hydrophilic group, acted as a swelling agent, further increasing water uptake and complementing chemical crosslinking processes (as discussed in section 7.4). Additionally, the inclusion of glycerol assisted in the PVA cryogelation process. Cryogelation refers to the use of low-temperature cycling that helps solidify homogeneous PVA liquid polymer solutions. A depression in the freezing point was observed in glycerol-containing solutions, allowing optimal crystallite formation with a controlled freezing rate (Du Toit & Pott, 2020). Glycerol also facilitated widespread crystallization between the PVA molecular chains, resulting in smaller crystallite sizes and greater transparency. This further enhanced the PVA gel’s diffusive properties and cell viability.

Throughout the experimental process, the team tested out the three organic cosolvents—glycerol, DMSO, and ethylene glycol. Tables 1, 2, 3 summarized the qualitative results of the PVA hydrogels with various organic cosolvent combinations and individual concentrations, along with the potential reasoning for notable successful and failed gelations.

Table 1. Qualitative Results of PVA Hydrogels with Various Organic Cosolvent Combinations| Date | Photo | Group Label | Qualitative Description of Hydrogel | Reasoning for Successful/Failed Gelation |
| --- | --- | --- | --- | --- |
| 2025/3/27 | ![](https://static.igem.wiki/teams/5624/result/result-tb1-1.webp) | Group #1  * 5% w/v PVA * No chemical additives * 15 min. autoclaving * FT | * Non-uniform crystallite formation * Brittle and hard * Opaque |  |
| 2025/3/27 | ![](https://static.igem.wiki/teams/5624/result/result-tb1-2.webp) | Group #2  * 5% w/v PVA * 50% v/v glycerol * 15 min. autoclaving * FT | * Uniform crystallite formation * Not brittle but hard * Relatively transparent | Semi-transparency and a yellowish tint arose from thermal degradation due to autoclaving and FT cycles. |
| 2025/4/10 | ![](https://static.igem.wiki/teams/5624/result/result-tb1-3.webp) | Group #3  * 5% w/v PVA * 80/20 w/w DMSO * 15 min. autoclaving * FT | * Uniform crystallite formation * Soft and demonstrated mechanical resilience * Fully transparent |  |
| 2025/4/10 | ![](https://static.igem.wiki/teams/5624/result/result-tb1-4.webp)(0 hour after immersion)![](https://static.igem.wiki/teams/5624/result/result-tb1-5.webp)(1 week after immersion) | Detoxification of Group #3, immersed in 99% ethanol. | * PVA hydrogel shrinkage * Failed water retention | Shrinkage due to the promotion of PVA physical crosslinking by ethanol. Alcohols encouraged hydrogen bonding between PVA chains, stiffening the network. |
| 2025/4/18 | ![](https://static.igem.wiki/teams/5624/result/result-tb1-6.webp) | Group #10  * 5% w/v PVA * 80/20 w/w DMSO * Heat plate slow-heating: 110°C + agitation * FT | * Improved dissolution and solution composition Note: Cell viability test performed by pipetting E. Coli onto the gel surface and immersing in BG-11 medium. Resulted in bacterial death. | Bacterial death was observed due to the nanoscopic pore sizes of the PVA hydrogel, which prevented *E. Coli* from penetrating and residing in the platform, thereby hindering survival. |
| 2025/6/13 | ![](https://static.igem.wiki/teams/5624/result/result-tb1-7.webp) | Group #12.1  * 3% w/v PVA * 50% v/v glycerol * Heat plate slow-heating: 110°C + agitation * FT | Failed gelation | Failed gelation due to exceptional freezing-point depression with ethylene glycol addition, where an even lower freezing temperature would be needed. |
| 2025/6/13 | ![](https://static.igem.wiki/teams/5624/result/result-tb4-1-1.webp)(before FT)![](https://static.igem.wiki/teams/5624/result/result-tb4-2.webp)(6/19, after FT) | Group #12.2  * 3% w/v PVA * 50% ethylene glycol * Heat plate slow-heating: 110°C + agitation * FT | * Uniform crystallite composition * Soft but fragile * Fully transparent | Ethylene glycol addition resulted in mechanically weak hydrogels, as it would form hydrogen bonds with PVA hydroxyl groups, reducing the number of PVA-PVA interactions and creating softer hydrogels with lower cohesive strength. |

Table 2. Qualitative Results of PVA Hydrogels with Various Glycerol Concentrations| Date | Photo | Group Label | Qualitative Description of Hydrogel | Reasoning for Successful/Failed Gelation |
| --- | --- | --- | --- | --- |
| 2024/4/10 | ![](https://static.igem.wiki/teams/5624/result/tb2-1.webp)(before FT)![](https://static.igem.wiki/teams/5624/result/result-tb2-2.webp)(after FT) | Group #6  * 5% w/v PVA * 40% v/v glycerol * 15 min. autoclaving * FT | * Uniform crystallite formation * Not brittle but hard * Yellow-colored and opaque | Semi-transparency and a yellowish tint arising from thermal degradation due to autoclaving and FT cycles. |
| 2024/4/10 | ![](https://static.igem.wiki/teams/5624/result/tb2-3.webp)(before FT)![](https://static.igem.wiki/teams/5624/result/result-tb2-4.webp)(after FT) | Group #7  * 5% w/v PVA * 60% v/v glycerol * 15 min. autoclaving * FT | * Uniform crystallite formation * Not brittle but hard * Yellow-colored and opaque | Semi-transparency and a yellowish tint arising from thermal degradation due to autoclaving and FT cycles. |

Table 3. Qualitative Results of PVA Hydrogels with Various DMSO Concentrations| Date | Photo | Group Label | Qualitative Description of Hydrogel | Reasoning for Successful/Failed Gelation |
| --- | --- | --- | --- | --- |
| 2025/4/17 | ![](https://static.igem.wiki/teams/5624/result/tb3-1.webp) | Group #9  * 5% w/v PVA * 70/30 w/w DMSO * 15 min. autoclaving * FT | Random error: Autoclaving resulted in non-uniform, highly viscous gelation with an immobile stirring bar.  → Failed gelation | Autoclaving consists of both steam and pressure, which could drive partial dehydration of the PVA hydroxyl (-OH) groups, pigmenting the polymer. Autocleaving was also a rather uneven heating method, resulting in uneven viscosity. |
| 2025/4/24 | ![](https://static.igem.wiki/teams/5624/result/result-tb3-2.webp)(Solidified PVA solution after poured into a rectangular mold) | Group #11  * 5% w/v PVA * 70/30 w/w DMSO * Heat plate slow-heating: 110°C + agitation * FT | * Uniform crystallite formation * Soft and demonstrated mechanical resilience * Fully transparent | Slow heating, with its gradual temperature increase, prevented thermal degradation and ensured uniform dissolution, thereby improving the control in crystallite formation and transparency. || 2025/5/1 | ![](https://static.igem.wiki/teams/5624/result/result-tb3-3.webp) | Detoxification of Group #11, immersed in 99% ethanol | * PVA hydrogel shrinkage * Failed water retention | Shrinkage due to the promotion of PVA physical crosslinking by ethanol. Alcohols encouraged hydrogen bonding between PVA chains, stiffening the network. |
| 2025/5/29 | ![](https://static.igem.wiki/teams/5624/result/result-tb3-4.webp) | Cell viability test performed by leaving pipette tips filled with *S. elongatus* PCC 7942 onto the gel surface of Group #11 and immersing in BG-11 medium. | Failed bacterial adherence and penetration | Resulted in bacterial death due to the nanoscopic pore size of the PVA hydrogel, which prevented *S. elongatus* PCC 7942 from penetrating and residing in the platform, thereby hindering survival. |

#### 7.3 Altering PVA Concentration

PVA concentration had a pivotal effect on determining the pore size of the hydrogels. According to past literature, an increased mass fraction (weight percent, wt% was used in the team’s experiments) would result in increased pore sizes (Sampath, 2016). A 10 wt% PVA demonstrated the most optimal mechanical properties with a dense structure, balanced porosity, and easy crystallization, though further increase in wt% would produce loose pores and fracture cracks (Chen et al., 2022). Table 4 summarizes the qualitative results of the PVA hydrogels with various PVA concentrations, along with the potential reasoning for notable successful and failed gelations.

Table 4. Qualitative Results of PVA Hydrogels with Various PVA Concentrations| Date | Photo | Group Label | Qualitative Description of Hydrogel | Reasoning for Successful/Failed Gelation |
| --- | --- | --- | --- | --- |
| 2025/4/10 | ![](https://static.igem.wiki/teams/5624/result/tb5-1-1.webp)(before FT)![](https://static.igem.wiki/teams/5624/result/result-tb5-1-2.webp)(after FT) | Group #4  * 15% w/v PVA * 50% v/v glycerol * 15 min. Autoclaving * FT | * Uniform crystallite formation * Not brittle but hard * Yellow-colored | Hard in texture due to the uneven heating resulting from autoclaving. A yellowish tint potentially arose from thermal degradation due to autoclaving and FT cycles. |
| 2025/4/10 | ![](https://static.igem.wiki/teams/5624/result/tb5-2.webp) | Group #5  * 25% w/v PVA * 50% v/v glycerol * 15 min. Autoclaving * FT | - PVA barely dissolved - Mixture was extremely viscous → Failed gelation | Autoclaving consists of both steam and pressure, which could drive partial dehydration of the PVA hydroxyl (-OH) groups, pigmenting the polymer. Autocleaving was also a rather uneven heating method. |
| 2025/4/17 | ![](https://static.igem.wiki/teams/5624/result/tb5-3.webp) | Group #17  * 8% w/v PVA * 80/20 w/w DMSO * 15 min. Autoclaving * FT | Random error: Autoclaving resulted in non-uniform, highly viscous gelation with an immobile stirring bar. → Failed gelation | Autoclaving consists of both steam and pressure, which could drive partial dehydration of the PVA hydroxyl (-OH) groups, pigmenting the polymer. Autocleaving was also a rather uneven heating method. |
| 2025/4/17 | ![](https://static.igem.wiki/teams/5624/result/tb5-4.webp) | Group #8  * 3% w/v PVA * 80/20 w/w DMSO * 15 min. Autoclaving * FT   Random error: Autoclaving resulted in non-uniform, highly viscous gelation with an immobile stirring bar. → Failed gelation | Autoclaving consists of both steam and pressure, which could drive partial dehydration of the PVA hydroxyl (-OH) groups, pigmenting the polymer. Autocleaving was also a rather uneven heating method. | |

#### 7.4 Glutaraldehyde as a Chemical Crosslinker

In PVA hydrogels, chemical crosslinking agents formed strong covalent bonds with the PVA hydroxyl groups. Chemical crosslinking typically reduced crystallization in PVA since covalent bonding hindered PVA chain mobility and its ability to form ordered crystalline domains, contributing to its exceptional transparency effects (Hassan & Peppas, 2018).

In our experiments, the chemical crosslinking agent used was a monoaldehyde—glutaraldehyde (GA). In the presence of optimal acidic catalysts (in our case, hydrochloric acid), glutaraldehyde could form acetal bridges between the pendant hydroxyl groups of the PVA chains (Hassan & Peppas, 2018). Tables 6 and 7 summarize the qualitative results of the PVA hydrogels with various glutaraldehyde concentrations and the finalized protocol, along with the potential reasoning for notable successful and failed gelations.

Table 5.Qualitative Results of PVA Hydrogels with Various Glutaraldehyde Concentrations| Date | Photo | Group Label | Qualitative Description of Hydrogel | Reasoning for Successful/Failed Gelation |
| --- | --- | --- | --- | --- |
| 2025/6/13 | ![](https://static.igem.wiki/teams/5624/result/result-tb6-1.webp) | Group #13  * 3% w/v PVA * 2.5 mL GA * 50% v/v ethylene glycol * Heat plate slow-heating: 110°C + agitation * FT | * Non-uniform crystallite formation * Brittle and hard * Opaque | Deviates from conventional principles regarding GA properties; group concluded as a manual, random error in the GA volume. |
| 2025/6/13 | ![](https://static.igem.wiki/teams/5624/result/result-tb6-2.webp)(after FT) | Group #14  * 3% w/v PVA * 5.0 mL GA * Heat plate slow-heating: 110°C + agitation * FT | Failed gelation | A failed gelation could arise from a pH too neutral or alkaline, as GA crosslinking requires acid catalysis. |
| 2025/6/13 | ![](https://static.igem.wiki/teams/5624/result/result-tb6-3.webp) | Group #15  * 3% w/v PVA * 7.5 mL GA * Heat plate slow-heating: 110°C + agitation * FT | * Non-uniform crystallite formation * Brittle and hard * Opaque | Failure should arise from over-crosslinking, or a GA volume too high. |
| 2025/6/13 | ![](https://static.igem.wiki/teams/5624/result/result-tb6-4.webp)(before FT)![](https://static.igem.wiki/teams/5624/result/result-tb6-5.webp)(after FT) | Group #16  * 3% w/v PVA * 12.5 mL GA * Heat plate slow-heating: 110°C + agitation * FT | * Uniform crystallite formation * Soft and demonstrated mechanical resilience * Fully transparent Note: After gelation, the hydrogel was immersed in tap water to determine the swelling ratio; failed water retention | The PVA hydrogel produced might have lost water due to thermal or chemical damage, specifically overheating (110°C), which resulted in chain scission and thereby poor retention. Crosslink success deviates from the expected chemical principles of GA; success concluded as the result of a random error in the added GA volume.Crosslink success deviates from the expected chemical principles of GA; success concluded as the result of a random error in the added GA volume |

Table 6.Qualitative Results of PVA Hydrogel Produced with the Final Optimized Protocol| Date | Photo | Group Label | Qualitative Description of Hydrogel | Reasoning for Successful/Failed Gelation |
| --- | --- | --- | --- | --- |
| 2025/9/12 | ![](https://static.igem.wiki/teams/5624/result/result-tb7-1.webp)(before incubation)![](https://static.igem.wiki/teams/5624/result/result-tb7-2.webp)(after incubation) | Group #18  * 6 wt% PVA * GA (0.1 mol GA/mol PVA -OH) * Heat plate slow-heating: 90°C + agitation * pH adjusted to 6.5 * Incubated for gelation | * Uniform crystallite formation * Soft and demonstrated mechanical resilience * Fully transparent | The key to successful gelation resided in acid catalysis, a more controlled heating temperature, and long-term incubation as a means of gelation. |

#### 7.5 Discussion

The team employed a comprehensive experimental process for PVA hydrogel production, focusing on modifying aspects including organic cosolvent addition and concentration, PVA concentration, and crosslinking methods to achieve the team’s desired hydrogel characteristics. Despite random errors (e.g., autoclaving as a failed heating method, erroneous additive volume), the team obtained qualitative results that generally coincided with the team’s research based on past literature.

Nevertheless, despite qualitative data on cell viability and visual observations, the team lacked quantitative analysis for PVA production due to limitations in device (e.g., tensile testers that could measure PVA hydrogel tensile strength in terms of Young’s Modulus) or intrinsic constraints (e.g., relative absorbance and therefore adhered cyanobacterial viability could not be determined due to hydrogel interference), offsetting the statistical significance and generalizability of the team’s results. One simple method that could be employed, however, was the calculation of swelling ratios, which could quantify the water retention properties of PVA hydrogels, accomplished through water immersion (Greene et al., 2023). The swelling ratio would be determined by the equation (Ws-Wd)/Wd, where Ws refers to the weight of the swelled hydrogel and Ws refers to the dry weight of the hydrogel.

Finally, although the team succeeded in optimizing PVA hydrogel transparency and tensile strength with the final protocol, the team failed in manipulating an adequate pore size for cyanobacteria to reside on the platform, given the negligible effects of PVA concentration on porosity. As a result, the team decided to draw an end to the PVA hydrogel production process and adopted a current feasible bioreactor model for carbon fixation.

### 8. Conclusion

Taken as a whole, the team’s experimental findings established a coherent understanding of inorganic carbon fixation and general functioning in cyanobacteria, *S. elongatus* PCC 7942. The CO2 assay verified continuous and quantifiable carbon fixation, while the stable O2 evolution (the process of producing O2 through oxygenic photosynthesis) and pH data confirmed active photosynthetic metabolism under physiologically balanced conditions. On the other hand, the precipitation assay revealed no detectable change in extracellular HCO3- concentration, indicating the absence of functional uptake HCO3- pathways via the outer membrane, supporting the team’s assumptions in the mathematical modeling.

Molecular analyses corroborated the team’s successful amplification and assembly of the *cmpAB* and *cmpCD* fusion constructs. Verification through bacterial transformation and PCR confirmed the readiness of the composite parts, ppsbA1-*cmpAB* and ppsbA1-*cmpC*D, for further use.

Through trial-and-error in modifying the crosslinking methods, PVA concentrations, and additive concentrations, the team produced a PVA hydrogel excelling in transparency and mechanical resilience, yet failed in creating quantitative justifications of the enhanced characteristics and creating adequate pore sizes, prompting the team to put the notion of an immobilized organismal adherence platform on hold.

Altogether, these outcomes verified the theoretical framework of the team’s experimental design, including dry and wet lab aspects until transformation. Future work will focus on validating the BCT1 transporter functionality post-transformation and will be discussed in the next section.

### 9. Future Directions

#### 9.1 Increasing Experimental Repeats for Statistical Reliability

Many of the team’s current experimental results were obtained from single or limited trials due to time and resource constraints. To ensure the reproducibility and statistical significance of the team’s findings, future work will include repeating key experiments multiple times under controlled and identical conditions.

#### 9.2 Future Experiments and Confirmations after Successful Cyanobacterial Transformation

Cyanobacterial transformation failures hindered the team’s progress throughout our project. We have applied various transformation methods, including chemical transformations, natural transformation, and electroporation, over several months. However, no colonies were observed throughout these attempts.

Due to such hindrances, there are several verification methods to conduct as a means of genetic construct and expression confirmation. After successful cyanobacterial transformation, 1% gel electrophoresis would be performed to verify the PCR products of the ppsbA1-*cmpAB* and *cmpCD* plasmids taken up by *S. elongatus* PCC 7942. Following genetic construct confirmation, the team would conduct RT-qPCR to detect mRNA induction levels of the *cmpAB* and *cmpCD* fusion genes, further accompanied by western blot analysis to assess the resulting fusion protein expression, together verifying the activity of the team’s engineered plasmids. The CO2 assay would also be performed on the engineered cyanobacteria that contained the team’s composite part designs, serving as a direct comparison with the wild type strain’s CO2 uptake efficiency. Data extracted from these test analyses could complement and further enhance the team’s predicted statistics for BCT1 overexpression, as determined using MATLAB mathematical modeling. The team’s fusion protein configuration, orientation, and functionality, modeled using Alphafold and DeepTMHMM, could also be confirmed.

#### 9.3 Using a Different Promoter for Maximal Protein Expression

To further enhance the expression of the BCT1 complex, an alternative promoter system can be considered aside from the ppsbA1 promoter that the team used. The Pcpc560 (phycocyanin operon promoter) is among the strongest constitutive and light-responsive promoters characterized in cyanobacteria (Zhou et al., 2014). Future comparative assays between the Pcpc560-driven constructs and the ppsbA1-based versions, evaluating mRNA and protein output through RT-qPCR and western blot analyses, would help determine the promoter’s effectiveness and optimize regulatory control for high-yield expression.

#### 9.4 Alternate PVA Hydrogel Applications as an Organismal Adherence Platform

Due to its transparency, mechanical resilience, and biocompatibility, PVA hydrogel can act as a stable support matrix for photosynthetic microorganisms, offering a controllable interface between biological activity and material structure. In the future, the team aims to develop a porogen leaching protocol to increase the pore size and overall porosity of the hydrogel, a fabrication method that uses porogen particles (e.g., sodium chloride) within the team’s PVA polymer solution (Sampath et al., 2016). By incorporating and subsequently dissolving and removing the porogens relative to PVA solidification, the hydrogel structure can be tuned to improve nutrient diffusion and organismal adherence. In addition, enhanced porosity is expected to promote more efficient cyanobacterial immobilization, improve mass transfer, and support the overall photosynthetic activity within the matrix, marking a potential big step toward replacing conventional bioreactors for carbon sequestration.

## References

Chen, Y., Li, J., Lu, J., Ding, M., & Chen, Y. (2022). Synthesis and properties of poly(vinyl alcohol) hydrogels with high strength and toughness. *Polymer Testing*, 108, 107516. https://doi.org/10.1016/j.polymertesting.2022.107516

Du Toit, J. P., & Pott, R. W. (2020). Transparent polyvinyl-alcohol cryogel as immobilisation matrix for continuous biohydrogen production by phototrophic bacteria. *Biotechnology for Biofuels*, 13(1), 105.

Hassan, C. M., & Peppas, N. A. (2018). Structure and applications of poly(vinyl alcohol) hydrogels produced by conventional crosslinking or by freezing/thawing methods. *Advances in Polymer Science*, 37–65. https://doi.org/10.1007/3-540-46414-x\_2

Ito-Miwa, K., Onoue, Y., Kondo, T., & Terauchi, K. (2025). Effect of ph on the cyanobacterial circadian oscillator in vitro. *Communications Biology*, 8(1). https://doi.org/10.1038/s42003-025-08273-8

Greene, C., Beaman, H. T., Stinfort, D., Ramezani, M., & Monroe, M. B. (2023). Antimicrobial PVA hydrogels with tunable mechanical properties and antimicrobial release profiles. *Journal of Functional Biomaterials*, 14(4), 234. https://doi.org/10.3390/jfb14040234

Sampath, U. G. T. M., Ching, Y. C., Chuah, C. H., Sabariah, J. J., & Lin, P.-C. (2016). Fabrication of Porous Materials from Natural/Synthetic Biopolymers and Their Composites. *Materials*, 9(12), 991. https://doi.org/10.3390/ma9120991

Zhou, J., Zhang, H., Meng, H (2014). Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria. *Sci Rep*, 4, 4500. https://doi.org/10.1038/srep04500

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