Design and construction towards a pan-microbial toolkit

Establishing genetic tractability in non-model microbes requires identifying genetic parts that function in a target host. However, the paucity and purported narrow host range of available parts means that successful identification is governed by serendipity. Instead, a more comprehensive and scalable process would be desirable. Here, we describe the design principles for a pan-microbial genetic toolkit in which phylogenetically-diverse parts can be assembled and tested for function in microbes using high-throughput readouts. The architecture is based on Golden Gate Assembly, which simplifies the addition of parts and the construction of combinatorial libraries. We used this framework to develop two modules: first, the POSSUM ( <underline>P</underline> lasmid <underline>O</underline> rigins and <underline>S</underline> election Marker <underline>S</underline> for <underline>U</underline> ndomesticated <underline>M</underline> icrobes) module for identification of replicating plasmids in non-model microbes which includes 29 plasmid origin of replication sequences, 23 selection markers, and 30 unique DNA sequences for tracking by sequencing; second, the MACKEREL ( <underline>M</underline> odular, NGS-tr <underline>ACK</underline> able <underline>E</underline> xp <underline>R</underline> ession <underline>EL</underline> ement) module, for identification of functional gene expression cassettes which includes 426 bacterial promoter-RBS sequences driving fluorescent reporter expression, trackable by flow cytometry. We demonstrate the use of these libraries to screen for functional promoter-RBS variants in 6 non-model microbes. Continued efforts to expand this pan-microbial toolbox will accelerate efforts to improve genetic tractability and guide research across the tree of life.