Parts Collection

Introduction

The construction of Biobricks via molecular cloning is a fundamental technique in synthetic biology and an essential component of iGEM projects. Recognizing the importance of synthetic biology skills during the COVID-19 pandemic period, Kang Chiao International School (KCIS) in New Taipei, Taiwan, established the Synthetic Bioresearch Laboratory in September 2021. The 2024–25 team represents the fourth cohort from KCIS Xiugang to participate in the iGEM competition.

From September to December 2024, team members underwent comprehensive training that covered a range of genetic engineering techniques, including polymerase chain reaction (PCR), primer design incorporating restriction enzyme sites, double enzyme digestion using NEB reagents, T4 DNA ligation, and bacterial transformation. Furthermore, the team members also developed proficiency in bioinformatics by utilizing databases such as NCBI to search for relevant publications and gene sequences.

Upon completing this initial phase, the class was divided into three groups, and each group was assigned to develop a project. These three teams subsequently presented their project proposals and were subjected to peer evaluation. Following a voting process in mid-December 2024, the final project was selected. During the winter break in January 2025, the team conducted a more in-depth literature review and formulated a comprehensive outline for the project.

In March 2025, following a complex thought process and difficulties in sourcing Synechococcus elongatus PCC 7942 from Taiwanese universities, the team officially commenced the project in April. The strain PCC 7942 was obtained and cultured at Dr. Ethan Lan’s laboratory within the Department of Biological Science and Technology at National Yang Ming Chiao Tung University (NYCU), where his lab kindly provided the team with a culture plate.

This year, the team’s inspiration stemmed from discussions in our biology course, which addressed the urgent need to mitigate rising atmospheric CO₂ levels and the broader global challenge of climate change. Taiwan’s substantial carbon emissions, primarily driven by urbanization and industrialization, motivated our group to explore a biologically based carbon capture strategy. Through an extensive literature review, we identified cyanobacteria as an ideal subject due to their innate role as photosynthetic carbon fixers and their potential as a scalable, sustainable platform. Our project aims to enhance the cyanobacterial CO₂-concentrating mechanism (CCM) by introducing the cmpABCD operon, which encodes high-affinity bicarbonate transporters, along with carboxysome-associated genes to improve carbon fixation efficiency. To further optimize regulation, we designed constructs under the control of a light-responsive promoter (ppsbA1), enabling synchronized expression with photosynthetic activity.

The team also studied works of previous iGEM teams from 2022 and 2024 to contextualize our project within ongoing synthetic biology efforts. For instance, the 2022 iGEM Rochester team developed CyanoVolt, a bio-photovoltaic system powered by engineered cyanobacteria that aimed to both capture CO₂ and generate electricity (Tan et al., 2022). While their work demonstrated the potential of cyanobacteria in climate-oriented applications, it emphasized energy production and bioproducts rather than optimization of the CCM itself. In contrast, our project has focused on directly improving the efficiency of carbon fixation by introducing the cmpABCD operon for bicarbonate uptake and reinforcing the carboxysome microcompartment in Synechococcus elongatus. Similarly, the 2024 iGEM ZJU-China team worked on enhancing carbonic anhydrase activity to accelerate the interconversion of CO₂ and bicarbonate. Unlike our design, which targets bicarbonate transport and carboxysome efficiency under light-regulated promoter, their strategy relied on extracellular CO₂ conversion and mineral deposition (Project Description | ZJU-China - iGEM 2022, n.d.). In summary, the project ideas from the preceding iGEM teams inspired our team’s overall experimental design, serving as a foundation upon which we established our own distinctive approach to enhancing the CCM in cyanobacteria.

Our final goal and design for the parts

Our project focuses on genetically engineering cyanobacteria to enhance carbon fixation efficiency by strengthening component 1: HCO₃^- uptake systems in the carbon-concentrating mechanism (CCM). We obtained Synechococcus elongatus PCC 7942 from Dr. Ethan Lan’s laboratory at National Yang Ming Chiao Tung University (NYCU).

To enhance the efficiency of HCO₃^- uptake, our team aims to overexpress the cmpABCD operon under the control of the light-dependent promoter ppsbA1, driving the expression of two fusion genes, cmpAB and cmpCD, rather than expressing each component separately. This approach is designed to accelerate the speed of complex formation, thereby facilitating faster HCO₃^- transport. The resulting polypeptides may facilitate the more rapid and stable assembly of the transporter complex, potentially improving the efficiency of HCO₃^- uptake in the engineered cyanobacterial system.

Through reading literature, the team discovered that adding a tag to the C-terminal of cmpB resulted in the loss of the permease’s function, and the cmpCD fusion gene with a mutation in cmpC’s regulatory component maximized bicarbonate intake. Without incorporating mutations, we eventually settled down on having cmpAB and cmpCD fusion genes to both maximize bicarbonate intake and protect our complex components without forfeiting protein detection via western blot (Rottet et al., 2024).

The method of employing genetically modified cyanobacteria to overexpress the cmpAB and cmpCD proteins for higher BCT1 complex formation allows the cyanobacteria to increase the uptake of HCO₃^-, thus increasing carbon fixation efficiency. By optimizing the expression and function of these fusion proteins, we expect to significantly increase inorganic carbon uptake in cyanobacteria, thereby boosting their photosynthetic efficiency and growth under carbon-limited conditions.

To justify the assembly and stability of the cmpAB and cmpCD fusion genes, the team performed AlphaFold, predicting the 3D protein structures of cmpAB and cmpCD fusion proteins via their amino acid sequences compared to cmpA, cmpB, cmpC, and cmpD alone, and also combined with the Deep TMHMM application to show that our fusion proteins, cmpAB and cmpCD, are at the correct orientations (detailed explanation below). After proving the stability of the cmpABCD when split into cmpAB and cmpCD fusion proteins, we moved on to construct the parts that include the light-dependent promoter ppsbA1 for overexpression.

The HCO₃^- Uptake Systems, BCT1 complex introduction

The team engineered two fusion genes, cmpAB and cmpCD, to overexpress the components necessary for assembling the high-affinity HCO₃^- transporter BCT1 complex. Each fusion protein contributes a specific function essential for the formation and activity of the BCT1 transporter, which transports HCO₃^- into the cytosol using energy from two ATPases contributed by cmpC and cmpD (Rottet et al., 2024) as described below (Fig.1):

cmpA (Solute-binding Protein, SBP, also called substrate-binding protein): cmpA is a solute-binding periplasmic protein facing the intermembrane space. cmpA has an active site that captures HCO₃^- and delivers it to the transporter complex cmpB. Structural studies have demonstrated that cmpA has a high affinity and specificity for HCO₃^-(Koropatkin et al., 2006; Q55106 · CMPB_SYNE7, n.d.).

cmpB (Transmembrane Permease): cmpB, a transmembrane protein, is a homodimer that facilitates the transport of HCO₃^- into the cytosol. To move ions against their concentration gradient using ATP energy, cmpB coordinates with cmpC and cmpD, the ATPase subunits, 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).

cmpC (ATPase with SBP): cmpC is an ATPase subunit in BCT1. It additionally functions as a substrate-binding protein (SBP). It hydrolyzes ATP through its N-terminal ATP-binding domain to supply the energy needed for HCO₃^- translocation into the cytoplasm. Meanwhile, its C-terminal substrate-binding domain helps the cmpB homodimeric channel optimize HCO₃^- transport (Price et al., 2008).

cmpD (ATPase subunit): cmpD, together with cmpC, forms the ATP-hydrolyzing motor that provides energy to the cmpB homodimeric channel, allowing for the active import of HCO₃^- into the cytoplasm in cyanobacteria. This ATP-hydrolyzing motor is pivotal to powering the movement of HCO₃^- against its concentration gradient. This enables the concentration of inorganic carbon within cyanobacteria for photosynthesis under low CO₂ conditions (Omata et al., 1999; Price et al., 2008).

Figure 1: The BCT1 complex contains the cmpA, cmpB, cmpC, and cmpD proteins

Revised Construct Design Using the ppsbA1 Promoter on pCOTS-pyl-GFP(35TAG) Plasmid

During the construction of the ppsbA1-cmpABand ppsbII-cmpCD expression plasmids, the team faced difficulties cloning the ppsbA1-cmpABconstruct. The team conducted a detailed analysis, including restriction enzyme digestion and sequence alignments. It revealed that the pAM1619 plasmid obtained from Addgene, which was supposed to contain the ppsbA1 promoter, did not have the correct promoter sequence. Additionally, it was found that the pCOTS-pyl-GFP(35TAG) plasmid, initially annotated as carrying the ppsbII sequence, actually contained the ppsbA1 sequence. To continue the project, the team revised its approach by using the ppsbA1 on the pCOTS-pyl-GFP(35TAG) plasmid to drive the expression of both the cmpABand cmpCD fusion genes, respectively, thereby enabling the construction of both genes under the same promoter in the pCOTS-pyl-GFP(35TAG) plasmid.

Basic part introduction

BBa_25BVILX0 (ppsbA1):

ppsbA1, a strong light-responsive promoter from the psbA operon, was selected to drive overexpression of the fusion genes (basic parts) cmpAB and cmpCD in cyanobacteria. In the current cyanobacteria species, Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, psbA genes encode the D1 reaction-center subunit of Photosystem II (PSII), whose rapid turnover under illumination makes psbA-family promoters among the most responsive and adjustable in cyanobacteria (Sacharz et al., 2009; Mulo et al., 2009). Notably, regulation of the ppsbA1 promoter is species-specific: in PCC 6803, ppsbA1 is regulated by light and inorganic carbon status; on the other hand, in PCC 7942, ppsbAI is active under low to moderate irradiation but becomes down-regulated under high light (Van der Staay et al., 1991; Nair et al., 2001). Utilizing the adjustability and flexibility of the ppsbA1 promoter allows us to achieve high and synchronized expression of the cmpAB and cmpCD fusion genes, supporting enhanced HCO₃^- uptake while fine-tuning cmpABCD expression under varying light and carbon conditions to maximize carbon fixation efficacy.

BBa_25SKOH98 (cmpAB):

The BCT1 protein complex, composed of the cmpA, cmpB, cmpC, and cmpD compartments, is located on the inner membrane of cyanobacteria, separating the periplasm from the cytoplasm. cmpA, a solute-binding protein (SBP) facing the periplasmic space, contains an active site that captures HCO₃^- and delivers it to the transporter complex cmpB. This transmembrane protein is a homodimer that facilitates HCO₃^- transport into the cytosol (Maeda et al., 2000). To promote the rate of HCO₃^- docking onto cmpA and thereby improve the efficiency of HCO₃^- transport through the cmpB transporter, the team constructed a cmpABfusion gene to shorten the time of cmpA and cmpB proteins assembly. This increased cmpABassembly rate allows faster transportation of HCO₃^- into the cytoplasm, optimizing the cyanobacterial CCM.

Furthermore, given the homodimeric nature of cmpB, the cmpABfusion protein allows for two cmpA compartments to exist in one engineered BCT1 complex relative to the single cmpA compartment in a native BCT1, increasing the amount of HCO₃^- moving into the cytoplasm with an increase in the number of SBPs (Rottet et al., 2024).

To justify the feasibility and applicability of the cmpABfusion protein, the team analyzed the 3D configuration of the cmpABfusion protein via AlphaFold, comparing it with the individual cmpA and cmpB assembly. According to the 3D protein configuration in Fig. 2, the cmpABfusion protein only presents a minor difference compared to the individual cmpA in conjunction with cmpB forming a complex. Most importantly, the HCO₃^- binding site at the N-terminus of the cmpA is not blocked in the cmpABfusion protein, indicating that cmpA’s core function, HCO₃^- transport, is preserved in the fusion protein.

Rottet et al.’s research on engineering the cyanobacterial bicarbonate transporter BCT1 illustrates that adding a tag to the C-terminus of cmpB causes the permease to lose function, however, the team designed the His-tag was at the N-terminus of cmpA (Rottet et al., 2024). Therefore, the team predicted that the cmpABfusion protein design is feasible for the project.

Figure 2: The 3D structures of the cmpABfusion protein were compared to the complex formed by individual cmpA and cmpB compartments, predicted with AlphaFold

BBa_25N38C54 (cmpCD):

In wild-type cyanobacteria, the native cmpC and cmpD ATPases complete the BCT1 assembly as ATP-hydrolyzing motors that provide energy, allowing the active import of HCO₃^- through the cmpB homodimeric channel into the cytoplasm (Omata et al., 1999; Price et al., 2008). After using Alphafold to verify the structural stability of the cmpCD fusion protein compared to the individual cmpC and cmpD protein compartments, we discovered that the fused and individual constructs showed visible differences in configuration (Fig. 3). Nevertheless, according to a functional analysis conducted by Rottet et al., a cyanobacterial cmpCD fusion protein design with a mutation of the cmpC regulatory component cloned to C3 plant chloroplasts demonstrated maximized HCO₃^- uptake out of the seven mutated genetic constructs tested, justifying the feasibility of our cmpCD fusion protein design (Rottet et al., 2024).

Figure 3: The 3D structures of the cmpCD fusion protein was compared to the complex formed by individual cmpC and cmpD compartments, predicted with AlphaFold

To conclude, the team engineered two fusion proteins, cmpAB and cmpCD, rather than expressing each component separately to enhance the speed of complex formation, increase the number of SBPs expressed, and improve HCO₃^- docking onto cmpA. Accompanied by the predicted configuration confirmations of the cmpAB and CD fusion proteins compared to the individual cmpA, B, C, and D compartments via Alphafold, we thereby hypothesize that the resulting polypeptides from the fusion genes may promote more rapid and stable assembly of the BCT1 transporter complex, potentially improving HCO₃^- uptake, CCM, and ultimately photosynthetic efficiency in the engineered cyanobacterial system.

Composite part introduction

BBa_25K6M3E8 (ppsbA1-cmpAB) :BBa_25BVILX0 (ppsbA1)+BBa_25SKOH98 (cmpAB)

The ppsbA1 promoter in S. elongatusPCC 7942 is strongly activated under normal (low to moderate) light intensities, but reduced under intense illumination (Van der Staay et al., 1991; Nair et al., 2001). Given ppsbA1’s adjustability to various light conditions, the team cloned the cmpAB fusion gene downstream of the ppsbA1 promoter to induce its expression under normal light intensities. The resulting cmpABfusion protein increases the rate of cmpA and cmpB assembly, facilitating a more rapid delivery of HCO₃^- into the cyanobacterial cytoplasm. Furthermore, since the cmpB transporter complex is a homodimer, the cmpABfusion protein would be able to accommodate two cmpA units (each functioning as an SBP that binds HCO₃^-), as opposed to just a single cmpA in the native BCT1 complex. This modification can thereby enhance the amount of HCO₃^- transported into the cytoplasm. The team created the ppsbA1-cmpAB construct using SnapGene (Fig. 4) and successfully cloned the construct, as demonstrated in the proof-of-concept experiments presented in the “Results” section.

Figure 4: Plasmid map of the ppsbA1-cmpAB construct with the His-tag, antibiotic resistance, and origin of replication labeled, created with SnapGene

BBa_25ZLGSPR (ppsbA1-cmpCD) : BBa_25BVILX0 (ppsbA1)+BBa_25N38C54(cmpCD)

The ppsbA1 promoter in S. elongatusPCC 7942 is highly active under low to moderate light, but demonstrates reduced activity under high light conditions (Van der Staay et al., 1991; Nair et al., 2001). With the ppsbA1 promoter’s adaptability to various light conditions, the team cloned the cmpCD fusion gene downstream of the ppsbA1 promoter to induce its expression under normal light levels. In cyanobacteria, the cmpC and D compartments complete the BCT1 assembly as crucial ATP-hydrolyzing motors that supply energy for HCO₃^- transport through the cmpB homodimeric channel into the cytoplasm (Omata et al., 1999; Price et al., 2008).

We analyzed the 3D protein structure of the cmpCD fusion protein using AlphaFold (Fig. 3) and compared it to the individual cmpC and cmpD compartments assembled. Although the configuration of the cmpCD fusion protein differs from that of the individual cmpC and cmpD subunits, a functional analysis conducted by Rottet's research team used a cmpCD fusion protein with a mutated cmpC regulatory component. When cloned into C3 plant chloroplasts, the mutated construct resulted in enhanced HCO₃^- uptake (Rottet et al., 2024), further supporting our cmpCD fusion gene design strategy. The team created the ppsbA1-cmpCD construct using SnapGene (Fig. 5) and successfully cloned the construct, as demonstrated in the proof-of-concept experiments presented in the “Results” section.

Figure 5: Plasmid map of the ppsbA1-cmpCD construct with the His-tag, antibiotic resistance, and origin of replication labeled, created with SnapGene