Multiplexing RNA Addressable Memory to Characterize Synthetic Circuits in Microbial Communities

Predicting and validating a synthetic circuit’s functionality within non-model microorganisms remains elusive and understudied in microbiome engineering. Although the development of intricate circuits in laboratory strains has become easier and more impressive over the years, application of these advancements into real communities and practical environments has fallen short. To better understand how synthetic parts function in microbes present within communities, we apply and advance a new molecular biology tool, RNA addressable memory (RAM). This tool records which microbial community members receive an engineered plasmid via conjugation. A trans-splicing reaction cleaves the 16S rRNA and attaches a synthetic barcode onto the freshly made edge of the rRNA, forming a chimeric RNA that contains species-specific information (16S) and evidence of horizontal gene transfer (barcode). The identity of transconjugants can easily be read-out using high-throughput amplicon sequencing. In short, RAM records who produced a transcriptional event. However, this technology is not limited to just studying horizontal gene transfer. Here, we extend the RAM technology to demonstrate an additional layer of information storage. We show that RAM can be used to store multiplexed information by changing the barcode, addressing synthetic biology questions of “What, Where, and How?’. Unique barcodes are paired with different synthetic parts and deployed in a microbe to understand and predict how the parts function in microbial communities.