Conclusion
Project PHAntom demonstrates a comprehensive synthetic biology approach combining metabolic rewiring and genetic regulation to enable programmable production of PHBV. The team successfully designed three plasmid constructs, pPHBV, pCo, and pSB1A3-sgRNA–SadCas9 (CRISPRi system), using standard iGEM BioBrick assembly techniques. PCR verification confirmed the expected sizes of all target inserts, and the cloning strategies utilized careful use of restriction enzymes, dephosphorylation, and ligation to minimize self-ligation.
At the metabolic level, the team strategically leveraged heterologous enzymes and native E. coli pathways to create a functional propionyl-CoA branch for 3-hydroxyvalerate (3HV) incorporation into PHBV. Genes encoding the enzymes H. Mediterranei bktb, S. flexneri TdcB and YciA, E.Coli TdcE, Y. pestis TesB, S.azorense alpha-carbonic anhydrase, and human ADCY10 SAC were chosen to optimize flux through both the central carbon metabolism and the PHBV biosynthesis pathway. The CRISPRi module, targeting citrate synthase via SadCas9 and sgRNA, was included to fine-tune intracellular fluxes, which redirects carbon toward PHBV production, highlighting the integration of genetic regulation with metabolic engineering.
Our modeling component translated this biological framework into a set of differential equations describing dynamic changes in biomass, acetate, and PHBV concentrations. Numerical simulations predicted an optimal PHB concentration of 158 mM at 36 hours, with ideal volume and feed rates determined to be 2.0 L and 0.09 g/L. Modeling results also highlighted several areas for further refinement, including experimental validation of enzyme kinetics, acetate toxicity thresholds, and the relationship between enzyme expression and acetate overflow. These findings provide a quantitative foundation for improving pathway efficiency and support iterative design cycles between computational predictions and experimental validation.
To supplement the project, we developed Spinova, a dual-axis random positioning clinostat machine that bridges the modeling and wet lab components by simulating non-earth gravitational conditions for bacterial culture. Additionally, FilaNova, a filament extruder designed to convert PHBV produced by engineered bacteria into usable 3D printing filament for various applications, was also developed. Spinova enables studies relevant to PHBV production under low-gravity environments, and it also represents a novel, adaptable platform for future space biology experiments. Initial mechanical challenges, including torque limitations and gear slippage, were addressed through design improvements. Future iterations will incorporate higher-torque motors to enhance stability and load capacity. This innovation aligns with NASA’s Artemis and Moon-to-Mars initiatives by offering an Earth-based tool to study how altered gravity affects microbial growth, gene regulation, and material production.
