Design and construction towards a pan-microbial toolkit
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Arcadia Science)
Abstract
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 ( P lasmid O rigins and S election Marker S for U ndomesticated M 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 ( M odular, NGS-tr ACK able E xp R ession EL 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.
Article activity feed
-
Glad to hear it!
This is a good point - can I ask if you're referring to a specific protocol (e.g. standard golden gate assemblies vs library golden gate assemblies) or just more generally?
As an aside, the authors of the Loop/uLoop Assembly systems posted some excellent resources on protocols.io, which I found very useful! (e.g. https://www.protocols.io/view/loop-and-uloop-assembly-14egn6rml5dy/v5)
-
Again, thanks for the feedback, makes sense.
I think the previous thread has some overlap with this one. Just to delve a little more, lots of the tools we have put together here have been reported to work across a range of bacterial phyla. Of course, that's not the same as demonstrating it in our hands directly! But broadly, we wanted to optimize for making these tools available (i.e. described and accessible).
-
Hi, thanks for the comment! We'll certainly bear this feedback in mind for edits/revisions to the manuscript.
To discuss in a little more detail, here we simply selected a set microbes for which we had previously obtained positive data from our ORI-marker screening approach (i.e. identified functional conjugation conditions and plasmids).
Having said that, the tools were generally designed to have functionality across a broader range of bacteria than those we test here. The awesome work of Yim et al. shows that the promoters they mined can work across at least three bacterial phyla, which was our rationale for selecting them. And the ORIs and markers we built are known work across a range of phyla too.
-
Data and Resource Availability
This is great that the plasmids are available through Addgene and the scripts/data on Github! I wonder if there is a general protocol that could be posted on Protocols.io for example to help make this method more approachable for others to try?
-
The design framework presented here enables a scalable method for establishing key initial footholds in genetic tractability in non-model microbes, and is extensible for a growing library of genetic parts and for complex hierarchical assemblies. Further development and broad application of this pan-microbial toolkit will accelerate our ability to study and engineer diverse microbes.
It might also be nice to provide some discussion as to how extensible currently this toolkit would be to non-model bacteria in other phyla besides Pseudomonadota? Or even just other species within this phyla that weren't tested? I think giving some rationale to why these species were chosen to begin with would be useful as well as how adaptable/usable are the given tools for diverse bacteria?
-
6 non-model microbes in pooled screens.
It might be nice to name the strains used here in the introduction so the reader has context earlier on for how taxonomically broad the toolkit works in. Context also for why these 6 strains were chosen for developing this toolkit would also be helpful - are they of biotechnology significance and highly desired for better tools?
-
