Beyond Model Organisms Plumbing the Extremes

[THEO] Okay, so almost every microbe you'd want to work with in a lab — the workhorses, the ones we know how to poke and prod — you could probably fit them on one page. E. coli, baker's yeast, a couple of Bacillus strains, maybe. That's it. That's the roster.

[DR. MARA] And the planet is running something closer to a trillion species. So when people say "non-model organism," what they really mean is "the ninety-nine-point-nine-nine percent we've never figured out how to grow, transform, or ask a question of."

[THEO] Right! And "model organism" isn't some deep biological category. It just means we got lucky, or worked really hard, and built the tools. You can get DNA into it, you can select for what you want, you can read the result. A model organism is basically an organism with good plumbing.

[DR. MARA] That's actually the honest definition. And the reason it matters — why this is a subject worth a whole segment — is that the interesting chemistry lives out in the wild. Organisms that eat methane, that fix nitrogen in the soil around plant roots, that survive in sea ice at temperatures where we'd expect metabolism to just stop. If you want a microbe to make a fuel, or a material, or clean something up, you often don't want to bolt that trait onto E. coli. You want to engineer the creature that already does it.

[THEO] So the whole game is: how do you turn a wild microbe into something you can actually engineer? And there are a few things you need. Mara, lay out the toolkit for someone who does, say, structural biology and hasn't touched a microbe since grad school.

[DR. MARA] Fair. First, you need to get DNA in. In bacteria and archaea that's transformation — chemically, or by electroporation, a quick electric pulse that opens the membrane — or conjugation, where a donor cell physically hands DNA over. Second, that DNA has to survive and replicate, so you need an origin of replication the organism recognizes, sitting on a plasmid, a little circular piece of DNA. Third, you need to control genes once they're in — turn them up, turn them down. And the whole time you're fighting the cell's immune system, restriction-modification, which chops up foreign DNA it doesn't recognize.

[THEO] That last one always gets me. You electroporate in your beautiful construct and the cell just goes "don't know you" and shreds it.

[DR. MARA] The microbial arms race, on your bench. So the story of this field is the story of building that plumbing for organisms that never had it.

[THEO] And I think the natural starting point is 2020, because you get two papers the same year coming at the problem from opposite ends. There's the Fatma review — bacteria, actinobacteria, cyanobacteria, yeast, fungi — making the argument that these things can be genuine cell factories if you build organism-specific tools. And CRISPR is the big lever there.

[DR. MARA] The framing I'd give: their claim is that intractability isn't a wall, it's a to-do list. Every organism is difficult in its own specific way, and you overcome it with tools built for that organism — often CRISPR-based, because CRISPR gives you programmable, targeted editing without needing the perfect classical genetics you'd have in a model strain.

[THEO] And then the other 2020 paper is, honestly, one of my favorite bits of science sociology. The marine protist study. Forty-one groups, over a hundred authors, funded by the Moore Foundation, and instead of everyone quietly failing on their own weird organism, they coordinated.

[DR. MARA] Thirty-nine species, spanning every eukaryotic supergroup. Protists, for the outside listener, are the single-celled eukaryotes — everything with a nucleus that isn't a plant, animal, or fungus. Wildly diverse, ecologically enormous in the ocean, and almost none of them had genetic tools.

[THEO] And they got thirteen first-ever transformation protocols out of it. First dinoflagellate nuclear transformation. First Antarctic diatom.

[DR. MARA] The turning point there wasn't any single protocol, though. It was that they published the failures. They wrote a synthetic roadmap — here's what we tried, here's what didn't work, here's how to approach a brand-new organism. That's the pivot from artisanal to systematic. From "my lab has a trick" to "here is a process anyone can follow."

[THEO] So 2020 sets up the two halves: build organism-specific tools, and build a general method for building them. And then the 2024 papers, to me, feel like the field growing up around that.

[DR. MARA] Take the In- and Out-Cloning work, de Vries and colleagues. This is deep in the plumbing. Modular cloning — MoClo, Golden Gate — is how you snap DNA parts together like standardized bricks, using enzymes that cut and leave short single-stranded overhangs that only fit the right neighbor. The problem is those overhangs leave scars, little leftover sequences, and every organism wants a different backbone.

[THEO] So picture LEGO, but the studs leave a smudge every time you connect two bricks, and also the baseplate is a different shape for every organism you care about.

[DR. MARA] That's close. Their contribution is using SapI, an enzyme that leaves a three-nucleotide overhang, so you can make the joins land on codon boundaries — scarless, in-frame. And the "Out-Cloning" half generates the organism-specific acceptor plasmid on demand. So it directly serves that 2020 goal: the tools stop being bespoke and become a parts catalog you assemble for whatever creature you've got.

[THEO] And then the Pseudomonas paper — the KT2440 CRISPRi one — that's the tool actually leaving the bench and going into dirt.

[DR. MARA] This is the one I'd flag as a real milestone. CRISPRi is CRISPR interference — you use a dead Cas9, dCas9, that can't cut, it just parks on a gene and blocks transcription. Turns genes down without editing them. They put a xylose-inducible dCas9 into the chromosome of Pseudomonas alloputida KT2440 using a mini-Tn7 transposon, which drops your cargo into a known safe site, and drove it with the pSEVA sgRNA parts.

[THEO] And the headline is they did it in the rhizosphere. Actual gene repression in the root zone, in situ.

[DR. MARA] First time, for in-situ rhizosphere repression. And they used it to knock down pyoverdine — a siderophore, an iron-scavenging molecule — and by disabling it they controlled how that microbe competes with its neighbors. That's the whole arc landing: an engineered non-model chassis doing something in its native environment, not a flask.

[THEO] Then there are the two that push on where the chassis can even survive. The polyextremophile framework, and Deming's piece on Colwellia.

[DR. MARA] The polyextremophile paper is a nice conceptual move. Instead of treating every extreme — heat, salt, radiation, cold — as its own problem, they collapse it to four proximal damage mechanisms: reactive oxygen species, covalent damage, protein unfolding, and membrane fluidity. And that tells you which extremes stack synergistically versus fight each other, so you can rationally design a strain for, say, space resource use.

[THEO] And Deming's Colwellia commentary is the reminder that biology's already out ahead of us — these psychrophiles in sea ice are exceeding the cold and metabolic limits we predicted.

[DR. MARA] Which is the honest closing note. The wild organisms keep outperforming the boundaries we draw for them. The whole field — from that protist roadmap to CRISPRi in the rhizosphere — is us catching up, building plumbing for creatures that were already doing the impossible.

[THEO] Creatures worth meeting. That's the whole show, really. More after the break.