A scalable framework for high-throughput identification of functional origins of replication in non-model bacteria
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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.
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Thank you for this comment, those are two excellent studies.
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The point about the host-to-host copy number variability is definitely an interesting one. There are many hand-waving points that could be made, but ultimately, we do not know the answer!
Thank you for the suggestion regarding Table 1.
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Yes that's correct, our brilliant culture team determined antibiotic susceptibility of these strains.
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Another great point. As you point out, in the species we tested here, none of the fungal ORIs would be expected to function. While they possess fungal ORIs, the two libraries we generated here would not be applicable in fungi because the antibiotic resistance cassettes possess bacterial expression elements. However, the Possum Toolkit contains commonly-used bacterial and fungal antibiotic resistance markers and we have used that feature to build libraries with fungal resistance markers.
In theory, you could even use the Possum Toolkit to build libraries with multiple resistance markers at once, each designed for different sets of organisms!
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This is a great point and I agree would be a really useful functionality!
The Possum Toolkit parts are all formatted according to the golden gate toolkit 'common syntax', so our antibiotic resistance gene parts will be cross-compatible with promoter parts from numerous existing golden gate toolkits for different species (e.g. CIDAR, the Fungal Toolkit, CyanoGate and more). On top of that we're actively working on generating (and sharing!) our own modular promoter library resources.
Thank you for this and all your comments!
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6 ORIs (32%) from eukaryotes
curious why fungal ORIs were constructed but not tested in fungi? would these plasmids work in fungi?
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the antibiotic resistance genes were not sufficiently expressed
a potential future step would be to include a promoter library in front of the resistance marker as well. is that already possible with the Possum toolkit?
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plasmid copy number
how well do you think plasmid copy number in the new hosts correlates to the original host? i'm wondering whether it would be useful to include any information about copy number in the original host in Table 1?
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organisms
it could be useful to mention other examples where people have tried related approaches to generating genetic toolkits. one example that comes to mind is the "Magic pools" paper: https://pubmed.ncbi.nlm.nih.gov/29359196/; another is here: Alker AT, Aspiras AE, Dunbar TL, Farrell M V., Fedoriouk A, Jones JE, et al. A modular plasmid toolkit applied in marine Proteobacteria reveals functional insights during bacteria-stimulated metamorphosis. bioRxiv 2023; 2023.01.31.526474.
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(Table 1,
This is a super useful table!
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pGL2_147 with a carbenicillin (CARB) marker and pGL2_150 with a gentamicin (GENT) marker
I'm curious why these two resistance markers were selected. Was it because of prior knowledge that the target strains selected are susceptible?
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