Results

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: 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-cmpAB(BBa_25K6M3E8) was successfully constructed.

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. 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: 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: 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 CO₃^2- 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: 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: 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: 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.

Date	Photo	Group Label	Qualitative Description of Hydrogel	Reasoning for Successful/Failed Gelation
2025/3/27		Group #1 5% w/v PVA No chemical additives 15 min. autoclaving FT	Non-uniform crystallite formation Brittle and hard Opaque
2025/3/27		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		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	(0 hour after immersion)(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		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		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	(before FT)(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.

Date	Photo	Group Label	Qualitative Description of Hydrogel	Reasoning for Successful/Failed Gelation
2024/4/10	(before FT)(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	(before FT)(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.

Date	Photo	Group Label	Qualitative Description of Hydrogel	Reasoning for Successful/Failed Gelation
2025/4/17		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	(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		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		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.

Date	Photo	Group Label	Qualitative Description of Hydrogel	Reasoning for Successful/Failed Gelation
2025/4/10	(before FT)(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		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		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		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.

Date	Photo	Group Label	Qualitative Description of Hydrogel	Reasoning for Successful/Failed Gelation
2025/6/13		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	(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		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	(before FT)(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

Date	Photo	Group Label	Qualitative Description of Hydrogel	Reasoning for Successful/Failed Gelation
2025/9/12	(before incubation)(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 (W_s-W_d)/W_d, where W_s refers to the weight of the swelled hydrogel and W_s 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-cmpCD, 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