Group 4: Minimally Adapted Carbon Sequestration Cell

Carbon dioxide in the atmosphere has reached a dangerous level for our planet, which has already spurred disastrous climate events, biodiversity loss, and threats to human livelihoods and homes. As a result, there is an urgent need to sequester this excess atmospheric carbon, though current technologies are either largely inefficient and/or extremely expensive. Inspired by naturally occurring carbon fixation cycles, such as the Calvin-Benson cycle in photosynthesis in plant cells, we leveraged principles from synthetic biology to develop a more efficient carbon fixation cycle and meet the urgent need we face. Using PCR, Gibson Assembly, and bacterial transformation, we constructed and implemented a fully synthetic carbon fixation pathway in vivo, where we inserted the POAP cycle into the JCVI Syn 3.0b minimal cell. To confirm the functionality of this pathway, we conducted gas chromatography-mass spectrometry analysis to characterize the change in metabolite levels relevant to the POAP pathway. However, multiple challenges, including factors such as acetate in the media, ATP limitation, ferredoxin availability, or protein instability at higher temperatures, complicate the interpretation of our results. Our work provides insight into the viability of using a carbon fixation pathway in vivo as a more efficient and cost-effective alternative to current carbon capture technologies. Future work on this project can address the current challenges in GC-MS analysis through further optimization, controlled testing conditions, and additional normalization strategies. Other future work may address how to manage oxalate, a byproduct of the POAP pathway, using a more complex and sturdier organism to host this pathway, and begin considerations for scaling.