The POSSUM Toolkit

ALL I NEED IS ONE GOOD PLASMID

Author: Nili Ostrov

Read the preprint (BioRxiv). Get the plasmids (Addgene).

Since starting Cultivarium, we’ve collected many “almost” stories from fellow microbiologists who ventured into exciting new microbes in their startup or thesis research and then dropped them in disappointment.

The number one reason for a promising microbe to be dropped is the lack of basic tools and protocols. And the absolute most basic tool - the one that can rescue an organism from oblivion - is a plasmid. 

Just one good plasmid.

It is hard to find a reliable plasmid that works in a new organism, one that’s not a relative of E. coli.

But how can that be? Surely one of thousands of available plasmids can work? Sadly, the most commonly used plasmids either originated from E. coli, engineered specifically for E. coli, or were only tested in E. coli. Good luck trying them all, one by one, to find the one that works in your marine/soil/alligator gut bacterium.

But even if you’re willing to try, it’s hard to know where to start. To get even one plasmid into one colony, you’ll need the right origin of replication, the right antibiotic selective marker and the right DNA delivery protocol - all at the same (!) time. 

Enter Cultivarium. 

Our mission is exactly that - to make non-model microbes more accessible by building tools that reduce the time, labor and cost of current bottlenecks. 

For example? 

Cultivarium’s first tool - Possum aka Plasmid Origins and Selectable Markers for Undomesticated Microbes (acronyms selected democratically by the team). 

The bottom line?

You can try this on any microbe you can grow in the lab. It will save you time and labor to find a plasmid that works in your organism because it tests many plasmids at the same time. 

How does it work? 

It’s a one-sample conjugation with a sequencing readout. 

Instead of trying each plasmid - each ORI and each marker in each organism - we took a combinatorial (multiplexing) approach; we’ve made a single sample to use. It’s a mix of many plasmids with many (barcoded) origins of replication. You deliver one sample (all plasmids at once) into your microbe. You collect all the colonies that survived and mix them into a single sample. You sequence this sample to find out which plasmids made it in. We also provide the collection of all genetic parts if you want to swap things in and out.

What organisms did you try so far?

In a single run of this assay in extremophile bacteria - salt-loving, high-pH, and some electrically active bacteria that have little protocols or tools - we found working plasmids for species where none were ever reported (Halomonas alkaliphila, Halomonas neptunia, and Shewanella electrodiphila) and came up with plasmids we didn’t know we can use in others (Duganella zoogloeoides, Pseudomonas alcaliphila, Shewanella oneidensis and Shewanella putrefaciens).

What do I need to get started?

To run our assay in your microbe, you’ll need the toolkit (we make it available through Addgene), our protocol (we make it open access on BioRxiv), and access to sequencing (that’s on you). The code is publicly available on GitHub. 

Big shout out to Charlie Gilbert, Stephanie Brumwell, Alex Crits-Cristoph, and Shinyoung Clair Kang!