Heterologous protein expression during deep space missions could be used for sensing, remediation, drug-delivery, and vaccines. Bacterial spores that can reside in a shelf-stable, ultra-light form factor without cold storage are desirable manufacturing candidates. This presentation focuses on comparing the performance of integrating vs. theta replicating plasmids for protein expression from germinated B. subtilis spores. Growth, and red fluorescent protein (RFP) expression profiles of 30 individual colonies were fit with a 4-parameter logistics and second order plus dead time models respectively to determine second order kinetic rate of expression (LKP) parameters for different cell and spore populations (average R2 of 0.998). RFP was regulated by a theophylline activated riboswitch to make the RFP readout highly sensitive to mutation effects. We observed 17X higher expression rates with theta replicating plasmids compared to integrating plasmids. However, this is at the expense of expression uniformity; we find 13X less homogenous expression from theta plasmids compared to integrating. We have determined that expression homogeneity is independent of plasmid stability, and, rather, relies on sequence fidelity and accessibility to Y-family polymerases. We observed that ΔPolYB mutants improve expression homogeneity almost 2X, while maintaining an expression rate that is 10X higher than the integrating plasmids. We also show that sporulation decreases protein expression homogeneity 2.7X. We conclude that theta replicating plasmids are better suited for protein expression from germinated endospores with B. subtilis Y-family polymerase knockouts. This work demonstrates that while spores have a potential for dormant cell factories, the heterogeneity of expression rate must be taken into account for sensor and vaccine applications.

Evaluating heterogeneity of protein expression from germinated Bacillus subtilis spores for shelf-stable cell factories in space

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.