Overview
For the 2025 iGEM project, our team aims to enhance the carbon fixation rate of cyanobacteria by overexpressing the cmpABCD operon, which encodes a high-affinity bicarbonate transporter —a component of the CO2-concentrating mechanism (CCM) —that facilitates the uptake of carbon dioxide in low-carbon dioxide environments. Our team has made significant contributions by adding new parts to the iGEM Parts Registry. This is achieved by splitting the cmpABCD into two fusion genes and placing them under the light-inducible promoter ppsbA1. As a result, we have achieved the two constructs: ppsbA1-cmpAB and ppsbA1-cmpCD to create a carbon fixation device capable of fixing carbon dioxide at a higher rate. For more information regarding the parts, please refer to the experiment and parts sections.
Parts Collection
Figure 1: cmpAB Fusion Sequence
cmpAB Fusion Sequence with His-tag (BBa_25SKOH98)
The part encompasses the cmpAB fusion sequence (a segment of the cmpABCD gene cluster) with a 6x His-tag added after the start codon (ATG). The addition of a hexahistidine (His-tag) allows for sensitive Western blot detection using anti-His antibodies or Ni-NTA conjugates, thereby eliminating the need for protein-specific antibodies. Since the His-tag is a short linear epitope that remains recognizable under denaturing SDS-PAGE conditions, it provides a consistent signal to the protein structure. The design choice for the part is based on its potential for cloning into plasmids using the ppsbA1 promoter. Furthermore, the design choice demonstrates high compatibility with BsrGI and Xbal restriction sites. The decision to split the cmpABCD gene into two parts (cmpAB and cmpCD) simplifies the cloning process for efficient gene expression because the original operon is too complex to express in cyanobacteria.. Furthermore, with the addition of the His-tag, the experimenter can undergo protein purification and detection.
Figure 2: cmpCD Fusion Sequence
cmpCD Fusion Sequence with His-Tag (BBa_25N38C54)
The part shares similarities with the cmpAB fusion sequence because it makes up the second segment of the cmpABCD gene cluster. The cmpCD fusion is cloned downstream of an existing His-tag in the ppsbA1 promoter plasmid, and the design choice is based on the use of Avrll and BamHI restriction sites. Adding an existing His-tag in the plasmid eliminates the need to include an additional start codon in the forward primer, making the cloning process more efficient. The ribosome will recognize the existing start codon in the plasmid, translate the His-tag, and continue translating the cmpCD as part of the same protein sequence. The fusion sequence is optimized through the ppsbA1 promoter plasmid, as the promoter exhibits light-inducible properties, resulting in transcription initiation in response to blue light.
Figure 3: ppsbA1 Promoter Plasmid and ppsbA1-cmpAB
Figure 4: ppsbA1 Promoter Plasmid and ppsbA1-cmpCD
ppsbA1 Promoter Plasmid and ppsbA1-cmpAB/ppsbA1-cmpCD
The PpsbA1 is a light-inducible promoter derived from cyanobacteria. The promoter activates in the presence of blue light, allowing the team to control the gene expression in a regulated manner. The plasmids containing the PpsbA1 promoter enable teams to utilize blue light as an inducer to activate gene expression at specific times, thereby reducing the need for other chemical inducers. With the plasmid, teams can conduct cloning experiments that require light-induced gene expression, especially in organisms that respond to light. A significant benefit of the PpsbA1 promoter is that it induces gene expression through blue fluorescent light. Other researchers can utilize this promoter for any function requiring on-off switching at specific times.
Furthermore, because these are naturally occurring promoters, other researchers will not be reliant on chemical inducers, which may produce adverse side effects. Finally, for other teams who wish to reduce the complications associated with photosynthetic organisms, the PpsbA1 promoter will assist in the transformation process as it has been adjusted to work with cyanobacteria, a photosynthetic organism. Therefore, the team inserts the ppsbA1 promoter before the cmp operon to manipulate its expression in response to light. By doing so, we aim to ensure consistent expression of the BCT1 bicarbonate transporter, thereby optimizing its performance under varying environmental conditions. This approach ensures that cyanobacteria can fix inorganic carbon under all CO2 levels.
Design Goal for synthetic biology design
Our team’s ultimate goal is to enhance carbon fixation efficiency in cyanobacteria by engineering the BCT1 transporter using synthetic fusion constructs. We designed two fusion genes, cmpAB and cmpCD, to accelerate and stabilize assembly of the BCT1 complex. We also placed these fusion constructs under the light-inducible psbA1 promoter to maintain expression under varying CO2 conditions. Lastly, to validate expression and activity, both fusion genes were also cloned with a His-tag for protein detection.
Beyond our own project and experiments, this design framework provides a generalizable approach for engineering multicomponent membrane transporters in prokaryotic systems. Future iGEM teams could adapt our fusion-gene design and light-inducible regulation strategies for other multicomponent transporters or metabolic pathways such as those in the ABC transporter superfamily, thereby ensuring efficient assembly and control within their chassis organisms. Besides enhancing carbon capture, our design links bacterial engineering with mechanical system development, serving as a valuable reference for future synthetic biology projects.
Hardware
Through the combination of the novel engineered cyanobacteria and BG-11 medium, we constructed a carbon fixation device, called CO2llectors. CO2 collectors are an improvement over other carbon fixation devices currently available on the market. When compared to other carbon-fixing technologies that do not utilize biological organisms, our cyanobacteria-powered device does not require additional energy, as cyanobacteria naturally fix carbon dioxide rather than relying on machinery. From a varying perspective, even when compared to other bioreactors, our strain has its advantages within the device because it is widely tested and has the potential to create biomass for a second revenue stream. Modifications to our prototype, from the initial design with the use of PVA gels and box-like containers to the final design, have made the device more effective and efficient when implemented in real-world applications, such as factories and power plants, because the engineered cyanobacteria have higher carbon fixation rates compared to other algae- or bacteria-powered carbon fixation devices. Moreover, the novel engineering of the enhanced cyanobacteria and BG-11 medium for maximizing cyanobacteria growth ensures a constant and reliable flow of carbon dioxide fixation.
Figure 5: Device Diagram
Modeling
Our team constructed a mathematical model to mimic and calculate the gradual change in ambient CO2 concentration driven by our genetically modified cyanobacteria's modified levels of carbon fixation. Moreover, it is specifically used in the simulation of the bicarbonate transport into a cell, Synechococcus elongatus PCC 7942’s carbon fixing mechanism, and integration of diffusion equations. Through calculations on these variables, the team is capable of establishing a comprehensive business plan, which can be applied to future projects and applications in industrial settings.
Education and Inclusivity
During this rousing iGEM project, our team's primary goal is not only to develop our experiment successfully but also to educate the public and spark thought on contemporary global issues. The team covers several facets of our project through education, such as informing the public about the general carbon fixation process of cyanobacteria and promoting ecological awareness. Comprehensive educational tools and systems are developed, including board games (Placeholder, Memory Match), G7-8 workshops, visually impaired lesson plans, storyboard creation, and Taitung camps. These educational projects are directed towards people aged 12-22, generally middle and high school students, university students, and other iGEM teams. Specifically, the Taitung camps focused on reaching rural indigenous communities and establishing a more inclusive learning platform on synthetic biology. We collaborated with the Taitung Bunan Summer Camp, which targeted indigenous youth in grades 5-6, aiming to create an educational experience that was meaningful to communities outside our own by simplifying complex ideas of DNA and inheritance into interactive lessons. Furthermore, we also established two board games that can enhance the spread of synthetic biology concepts: Memory Matching Game and Placeholder. All in all, our iGEM team establishes an academic program that raises awareness among the public, addressing the concept of sustainability and introducing synthetic biology to the general public.