A Ribozyme-Based Circuit for Gene Expression Tracking in Non-Model Organisms

Genetic circuits are robust tools for gene expression and have revolutionized our understanding of biological memory and signal actuation. Many of these systems have been developed and confined to model organisms whose Open Reading Frames (ORFs) are well quantified. For emerging areas of microbiome research such as the gut, wastewater, and soil, these circuits are impractical in non-model organisms whose transcriptional components are unknown or poorly understood. However, mRNA serves as an ideal actuator for biological memory, as accessing its information requires no knowledge of ORFs. Tools for manipulating mRNA to produce a measurable output are lacking but have potential to alleviate constraints on host organism for in vivo monitoring. Here, we present a simple circuit that uses a programmable guide sequence to direct a trans-splicing ribozyme to convert mRNA into a non-destructive and reversible electrochemical readout. To validate our system, we designed a complementation assay using E. coli EW11, a strain incapable of metabolizing inorganic sulfur. We programmed the ribozyme to target mRFP mRNA and output CysI, the hemoprotein subunit of sulfide reductase, enabling production of S^2- and survival in minimal media with sulfite as its only sulfur source. Furthermore, bioreactor growth assays using chronoamperometry could discern a S^2- readout for over 60 hours. Future research will center on targeting chromosomal genes in non-model organisms as well as develop new parallelizable circuits for synthesis of other redox biomolecules. We hope that this tool can be employed as a novel tool for improved study of microbial systems in situ.