POSSUM Unlocks the Biological Universe with Speed

What used to take years can now be completed in days.

At Cultivarium, we’re building tools to help scientists move fast with non-model microbes. These are the wild, wonderful organisms that haven’t benefited from decades of research and toolkits behind them, like E. coli or S. cerevisiae. But they might be faster, tougher, or better suited for the job you have in mind.

Our latest proof point? A rapid survey (BioRxiv preprint) of plasmids for Vibrio natriegens, the fastest-growing free-living organism. However, this story begins much earlier, with a profound passion for organisms that many scientists have overlooked.

The Microbes That Time Forgot

While the scientific world has spent decades perfecting E. coli and S. cerevisiae, we've been captivated by the vast microbial universe that remains largely unexplored. These "non-model" organisms—the bacteria, archaea, and fungi that are not typically listed in textbooks—often possess extraordinary capabilities that could revolutionize biotechnology.

The challenge? Working with non-model microbes is notoriously difficult. Scientists need genetic tools, such as plasmids, promoters, and origins of replication, that function reliably in their organism of choice. For well-studied microbes like E. coli, these tools have been refined over the course of several decades. For everything else, researchers often start from scratch, spending months or years just to get basic genetic manipulation working.

This is exactly the barrier we've dedicated Cultivarium to breaking down.

Vnat: Enter the Speed Demon

We’ve been in love with Vibrio natriegens (affectionately, Vnat) for nearly a decade. What makes V. natriegens special? Pure speed. This bacterium doubles every 10 minutes under optimal conditions, twice as fast as E. coli. That might not sound revolutionary until you consider the cumulative impact: if every biology lab in the world could cut its experimental timelines in half, the acceleration in scientific discovery would be staggering.

Back in 2012, when the Cultivarium team was just a couple of postdocs at Harvard, it took four years to establish foundational resources for Vnat (2016): the closed genome, delivery and molecular tools, and a roadmap to whole-genome functional genetics using CRISPRi. Why did we do all that? We believe that Vnat could accelerate science itself, and we wanted to share this as soon as possible. It would take us another 3 years (2019) to scale up the initial work and publish it in a journal (most of the time was spent here).

We weren't the only ones who recognized this potential. This microbe attracted attention from major players, including Synthetic Genomics Institute, where Matt Weinstock (now CTO of Novel Bio) developed a commercial version called Vmax. Matt's team has now launched CyClone, a commercialized strain designed to be even more user-friendly.

Can Vnat really be the new E.coli? That vision is now closer to materializing, and we would like to help bring it to fruition.

The Switching Cost Problem

But here's the catch that's holding back widespread adoption of any new microbial host: switching costs. Most scientists have invested years perfecting their experimental workflows around specific plasmids and genetic parts. They're understandably reluctant to abandon their tried-and-true setup for a new organism unless they're confident it will work with their existing toolkit.

This creates a chicken-and-egg problem. Scientists won't upgrade to other organisms without compatible genetic tools, but tool development relies on usage to drive innovation. Someone needs to systematically determine what works and implement it quickly enough to accelerate, rather than hinder, research progress.

POSSUM: A Parallel Approach

Enter Cultivarium’s POSSUM: Plasmid Origins and Selectable Markers for Undomesticated Microbes. It's a mouthful, but it works.

Instead of testing plasmids one at a time, we create a library of them, each with a different origin of replication (ORI) and antibiotic resistance markers, and introduce them all at once. Then, we sequence all surviving colonies together to read out which plasmid “survived” in the microbe. The surviving plasmids tell us which ORI and antibiotic work. This experiment is one complex DNA sample going into a microbe (all plasmids together, including an internal control), and one DNA sample loaded on the sequencer at the end (DNA from all colonies together, including the internal control).

It’s like casting a net of options and seeing which ones come back with a catch.

CyClone Meets POSSUM

CyClone is a new lab strain of Vnat being commercialized by Novel Bio. It’s designed to produce plasmid DNA quickly, which can be valuable for applications ranging from basic research to the manufacturing of therapeutics.

But switching your cloning system from E. coli to Vnat isn’t trivial. Could you use the same plasmids? The same antibiotic selection markers? What concentration of antibiotics should you use? Answering those questions used to be a slow process. When we worked on the original Vnat strain, it took nine months to build and test, one by one, just a small fraction of the plasmids we have in POSSUM. We need faster and more comprehensive answers.

We applied POSSUM to wildtype and CyClone Vnat strains. We treated CyClone like any unknown microbe. No assumptions. We ran the assay and got a clear picture of the working parts and protocol in under a week. Origins of replication, antibiotic resistances, it was all there. Our POSSUM results matched nicely with what was previously published about Vnat and extended upon what Novel Bio has set in their user manual.

The key value isn't only the speed of the assay, it's the confidence derived from the outputs. POSSUM lowers the switching cost barrier by building trust with scientists who might otherwise hesitate to switch to a new system. It provides researchers with a data-backed menu of what works.

The Bigger Picture

The CyClone project represents something larger than just characterizing one organism. It's a demonstration of how systematic, high-throughput approaches can accelerate the adoption of any non-model microbe. What might appear to be a trickle of testing individual organisms is part of our larger effort to explore the vast and largely unexplored microbial universe.

Every time we find a microbe with interesting properties, whether it's a bacterium that can degrade plastic, a fungus that produces a novel chemotherapy, or an archaea that is the most radiation-resistant organism, we face the same fundamental questions. How can we manipulate their genomes to learn from or harness them? The only thing holding us back is the time and effort required to develop tools for each of these organisms.

POSSUM and similar technologies can compress this timeline from years to weeks. It's infrastructure for a future where scientists work with the best microbe for the job, not just the best-studied microbe. The future of biotechnology isn't just about the few well-studied model organisms; it's about systematically exploring the entire tree of life.

Take a look at the BioRxiv preprint for the scientific details. And, if you’re working with a new microbe and are unsure where to start, let’s talk. Reach out to me and the team at info@cultivarium.org.

Read more about POSSUM
📜 BioRxiv preprint
📣 Cultivarium blog post

Learn about CyClone
🧬CyClone product overview