A scalable framework for high-throughput identification of functional origins of replication in non-model bacteria

Abstract

Microbial genetic manipulation requires access to engineerable plasmids that can be programmed to perturb genes, pathways and genomes. The extensive repertoire of plasmids available for model microbes, such as Escherichia coli, has facilitated fundamental biology studies and synthetic biology applications. However, the scarcity of plasmids for non-model microbes hinders efforts to broaden our biological knowledge and constrains the development of biotechnological solutions. In this study, we introduce a molecular toolkit and multiplexed screen to evaluate functional plasmids in non-model microbes. We constructed a collection of genetic parts consisting of 22 origins of replication (ORIs), 20 antibiotic selectable markers, and 30 molecular barcodes, which can be assembled combinatorially to create a library of plasmids trackable by next-generation DNA sequencing. We demonstrate our approach by delivering a pooled library of 22 ORIs to 12 bacterial species including extremophiles, electroactive bacteria and bioproduction strains. We report, for the first time, DNA delivery by conjugation and functional ORIs for Halomonas alkaliphila, Halomonas neptunia, and Shewanella electrodiphila. Furthermore, we expand the list of functional ORIs for Duganella zoogloeoides, Pseudomonas alcaliphila, Shewanella oneidensis and Shewanella putrefaciens. This screen provides a scalable high-throughput system to rapidly build and identify functional plasmids to establish genetic tractability in non-model microbes.

Design and construction towards a pan-microbial toolkit

“…Library members for which representation is reduced will be detected with lower sensitivity in ORI-marker screens, as the amount of DNA delivered will be decreased. However, we previously demonstrated detection of functional ORIs which made up as little as 0.11% of the total library 8 .…”

“…This reporter gene cassette was then assembled with either of two broad host range plasmid ORIs (pBBR1 and RSF1010), a gentamicin resistance cassette and conjugation oriT sequence ( Figure 4A and Supplementary Figure 11 ). Notably, we had previously determined the gentamicin resistance cassette and plasmid ORIs used here were functional in these species using our ORI-marker screen approach 8 . Since Golden Gate assembly transformations usually contain a non-negligible proportion of re-ligated destination vector, we created a set of 20 destination vectors in which the dropout part contained a ccdB cassette ( Supplementary Table 2 ).…”

“…The intended purpose of ORI-marker libraries is to provide researchers with a single, comprehensive resource to identify replicating plasmids in target microbes. To this end, we have previously described an experimental screen to identify functional plasmids from ORI-marker libraries in non-model microbes through pooled library screening 8 .…”

“…To generate pooled ORI-marker libraries, Level 1 barcoded-ORI plasmids were first manually pooled by combining equal volumes of 50 nM plasmid stocks, with the exception of the dummy ORI plasmid (pGL1_46) for which double the volume was used. Since the fold enrichment in NGS reads mapping to each ORI compared to the dummy ORI is used to assign functional ORIs in the ORI-marker screen 8 , maintaining the dummy ORI at a relatively high abundance in ORI-marker libraries helps reduce the risk of false positives. Next, SapI Golden Gate assemblies were performed as described in the previous section treating the pooled barcoded-ORI as a typical assembly part.…”

“…One promising avenue to achieving this is the development of widely-accessible resources that provide: (1) highly diverse, combinatorial libraries of genetic parts compatible with a broad range of microbes, and (2) validated high-throughput screening methods to rapidly identify functional genetic parts in target microbes (Figure 1). This design can dramatically simplify the process of identifying functional genetic parts in non-model microbes, as exemplified in several recent studies [1][2][3][4][5][6][7][8] .…”