Biology subjects

Dysart, M. J.

Publications and source records attributed to Dysart, M. J..

3 recordsLinked to original sources

bioRxiv2026

Storing >1 byte of information in 16S ribosomal RNA using orthogonal trans-splicing ribozymes

TEXT ABSTRACTCatalytic-RNA (cat-RNA) expressed from mobile DNA can record cellular events, such as the uptake of plasmids via horizontal gene transfer, by splicing a barcode onto 16S ribosomal RNA (rRNA) - a system termed RNA addressable modification (RAM). However, scaling RAM to record multiple simultaneous biological events requires large numbers of orthogonal cat-RNA whose signals reflect the biological features under investigation rather than variability arising from the barcode sequence. Here, we explore how to design orthogonal cat-RNA to record information about multiple plasmid-encoded traits in parallel. We show that cat-RNA having tRNA-derived barcodes with sequence variation in the anticodon stem-loop present greater signal consistency within Escherichia coli than mRNA-derived barcodes. When orthogonal cat-RNA designs harboring tRNA-derived barcodes were evaluated in Vibrio natriegens and Pseudomonas putida, increased variance was observed compared with Escherichia coli. Nevertheless, the signal consistency was sufficient to use these orthogonal cat-RNAs to report on the relative activities of four promoters and two origins of replication by sequencing barcoded-rRNA derived from the three organisms. These results show how RAM can be multiplexed to report on mobile DNA features in microbial communities and illustrate the importance of accounting for variability in RNA outputs when designing and interpreting multiplexed RNA barcoding data. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/738544v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1f6d62aorg.highwire.dtl.DTLVardef@6696c6org.highwire.dtl.DTLVardef@134aeb4org.highwire.dtl.DTLVardef@1b9ceeb_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Cross-order detection of bacteriophage transduction in communities using ribosomal RNA barcoding

Bacteriophages (phages) facilitate gene transfer and microbial evolution in all ecosystems and have applications as tools for engineering microbiomes and as antimicrobials. Historic efforts to map phage hosts, such as plaque assays, are limited to culturable bacteria, are low throughput, and are hard to apply in environmentally-relevant contexts. To overcome these limitations, a synthetic ribozyme that stores information about DNA uptake in 16S ribosomal RNA (rRNA) was used to identify phage-host interactions by integrating the ribozyme into phage-plasmid P1 and performing targeted 16S rRNA sequencing following transduction. Experiments in synthetic and wastewater communities revealed Aeromonadales as a novel P1 host order and transduction of P1 into pathogens. Host range varied across phagemids with different origins of replication and phage particles with different tail fibers. This work shows how autonomous barcoding can be used in phages to identify the molecular controls on their host range in communities.

synthetic biology↗

bioRxiv2023

Information storage across a microbial community using universal RNA memory

Biological recorders can code information in DNA, but they remain challenging to apply in complex microbial communities. To program microbiome information storage, a synthetic catalytic RNA (cat-RNA) was used to write information in ribosomal RNA (rRNA) about gene transfer host range. By reading out native and modified rRNA using amplicon sequencing, we find that 140 out of 279 wastewater microbial community members from twenty taxonomic orders participate in conjugation and observe differences in information storage across amplicon sequence variants. Twenty of the variants were only observed in modified rRNA amplicons, illustrating information storage sensitivity. This autonomous and reversible RNA-addressable memory (RAM) will enable biosurveillance and microbiome engineering across diverse ecological settings and studies of environmental controls on gene transfer and cellular uptake of extracellular materials. One-Sentence SummaryRibosomal RNA sequencing detects cellular events recorded across a wastewater microbial community using synthetic biology.

synthetic biology↗