Rational engineering enhances the signal and modularity of an RNA barcoding technology to track gene transfer in microbiomes
Year: 2026
Type: preprint
Abstract: ABSTRACT Horizontal gene transfer drives microbial evolution and offers a powerful strategy for precision microbiome engineering. To track gene transfer within complex communities, we previously developed RNA-Addressable Modification (RAM), an RNA-barcoding technology where a mobile catalytic RNA barcodes host 16S ribosomal RNA (rRNA) upon gene transfer. However, the first-generation RAM suffered from low barcoding efficiency and lacked modularity, limiting its sensitivity and versatility. Here, we present RAM v2, a re-engineered system with significantly enhanced performance and modularity. By incorporating natural ribozyme structural motifs and improved barcode stability, we achieved a ∼200-fold increase in barcoded rRNA signal. To enhance modularity, we integrated CRISPRi-based repression and ribozyme insulators, facilitating easy promoter swapping. We validated RAM v2 on a mobilisable plasmid delivered to a complex wastewater microbial community, demonstrating a substantial increase in signal over the original system while barcoding similar taxa. These improvements enable higher-resolution, more sensitive monitoring of horizontal gene transfer, providing a robust toolkit for accelerating the study of gene transfer in microbial communities and advancing targeted microbiome engineering.
Institution: Rice University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G122402259, CAREER: Harnessing horizontal gene transfer to engineer environmental microbiomes in situ, Tools4Cells: Harnessing the plasmidome for environmental engineering of microbial communities using synthetic biology, CAREER: Selective activation of catalytic RNA to control energy flow in microbial consortia
OpenAlex ID: W7155210348