Experiment

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. 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. elongatusPCC6803 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. 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

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 to verify the PCR and gene expression success.

Double-enzyme Digestion

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

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

The ligated ppsbA1-cmpAB and ppsbA1-cmpCD plasmids were transformed into DH5α competent bacterial cells after bacterial plasmid extraction 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

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

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 CO₃^2- 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

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

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 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. elongatusPCC 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. 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 psbA1-cmpAB and psbA1-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

References