Poster U16 - Development of an In Vivo Carbon Fixation System in the Syn3B Minimal Cell

Introduction: The need for an efficient, self-sustaining system of carbon sequestration is increasingly evident due to the rapid accumulation of carbon dioxide in earth's atmosphere. A recent study established the in vitro functionality of a synthetic carbon fixation cycle containing four enzymes, titled the POAP pathway (Figure 1). We propose to demonstrate the functionality of the POAP pathway in vivo, using the JCVI Syn3B Minimal Cell which was evolved using Thermal Adaptive Lab Evolution (TALE) to grow at ~44°C (from its original temperature of 37ºC) so that it can survive the optimal operating temperature for the pathway enzymes. To achieve this, we developed two 2-Enzyme Plasmid Designs, allowing us to create minimal cell strains that encode half of the POAP pathway each, which will then be pieced together to build the full cycle. Ongoing work focuses on testing the efficacy of these strains in processing either acetate or pyruvate and outputting the opposite using Gas Chromatography-Mass Spectrometry (GC-MS). We anticipate our findings will be a starting point for using the minimal cell as a screening platform for metabolic processes such as a minimal carbon sequestration system. Our results lay the foundation for future work optimizing the POAP pathway in vivo and present a novel understanding of enzymatic carbon sequestration systems.

Materials and

Methods: The four enzymes of the POAP pathway were first screened using the BRENDA database for optimal genes, then inserted into Escherichia coli for assembly (Figure 2). The plasmids from the transformed E. coli were then purified and evolved minimal cell strains were transformed with the assembled single enzyme constructs. To build the 2-Enzyme design, the smaller insert genes (ACS and OAH) had to be implemented into the already constructed single-enzyme backbone (PFOR and PYC, respectively). Homologous regions corresponding to the respective backbones of each insert were built up to 50 base pairs for the upstream region and 200 base pairs for the downstream region using sequential cycles of PCR and different forward primers. The backbones were linearized using enzyme digestion, HindIII for PYC and EcoRI for PFOR. The pieces were then bound together using Gibson Assembly and transformed into DH5-alpha E. coli cells for amplification, all of which proved successful using Plasmidsaurus sequencing. Next steps involve the transformation of these plasmids into the minimal cell. This will be followed by performing analysis on the novel 2-Enzyme strains in order to determine whether the product does accurately fit the specifications and constraints of the minimal cell design. GC-MS will be used to quantify successful enzyme activity including the perturbations of the substrate and product levels between the original and transformed strains (Figure 4).

Results, Conclusions, and Discussions: Since this project is in its early stages, we spent extensive time troubleshooting the cloning steps involved in engineering the two 2-Enzyme plasmids (Figure 4). The main obstacles were building up the large overhangs onto the insert genes for OAH and ACS, and attempting to assemble each insert into their respective backbones using E. coli Assembly. To tackle the first problem, we changed the reverse primer we were using for PCR, optimizing it to the binding site between the backbone and insert. Each PCR run would allow a short sequence of new nucleotides matching the backbone to be added to the upstream end of the genetic fragment. ACS required 2 consecutive PCR runs while OAH required 7, and we saw success using gel electrophoresis which showed the bands getting larger after each reaction until it reached the expected band size (Figure 5). To solve the second problem, we switched from using E. coli Assembly to Gibson Assembly, which is known to be far more efficient, and our sequencing results confirmed we got the correct gene pairs for our pathway. The most recent result is our first successful transformation of the minimal cell with the OAH-PYC and ACS-PFOR 2-Enzyme Plasmids which was confirmed through Plasmidsaurus. Analysis with GC-MS will provide insight into whether these enzymes are working together as intended, and the desired metabolic perturbations will give us the verification needed to start the full pathway assembly. In conclusion, since the initial development of the POAP pathway, we are one of the first few groups to test a minimized carbon fixation cycle in-vivo. We have developed a pipeline to engineer a full metabolic pathway into a viable organism from the ground up. By using the minimal cell as our biological chassis, we demonstrate its efficacy as a platform to screen and analyze the effects of engineered proteins on basic biological functions. Additionally, our application of the ALE technique to grow a thermo-resistant strain of the minimal cell serves as an example for the evolution of thermo-resistant strains of other single-celled organisms.

Acknowledgements (Optional): This work was done as part of the Senior Design Project at the Shu Chien-Gene Lay Department of Bioengineering at the University of California, San Diego in collaboration with the J. Craig Venter Institute. We would like to thank our mentor Dr. Suzuki for his continued support, as well as the assistance of the Synthetic Biology group at the J. Craig Venter Institute, including Dr. John Glass and Dr. Ayako Murao. We would also like to express our gratitude to our Senior Design Professor, Dr. Alyssa Taylor, and our advisors Yan Zhe Liu, Andrew Nguyen, Neel Dhar, Aishwarya Mitra, and Amit Klein for their guidance throughout this project.