Beyond Model Organisms Engineering

[THEO] Okay, picture the microbial world as a casting call. For decades, molecular biology only ever hired two actors — E. coli and baker's yeast. Every scene, every movie, same two faces.

[DR. MARA] Which is understandable. We knew how to grow them, how to get DNA into them, how to make them behave. That familiarity is exactly why they became the standards.

[THEO] Right, but there are millions of other microbes out there — bacteria that live in sea ice, cyanobacteria that shrug off salt, fungi that make compounds we can't synthesize — and almost none of them were on the payroll. That's the story today. Why the field started saying, hang on, some of these weirdos are worth meeting.

[DR. MARA] And "worth meeting" is doing real work in that sentence. A non-model organism might already make the exact molecule you want, or survive the exact condition your process needs. The problem was never biology. It was the toolkit.

[THEO] So define the villain for me. What makes an organism "intractable"?

[DR. MARA] Several things, and they compound. First, you often can't get DNA in — a bacterium may chew up foreign DNA with its restriction-modification system, the molecular immune system that recognizes and cuts sequences it doesn't recognize as self. Second, even if the DNA gets in, your plasmid needs an origin of replication that actually fires in that host, or it won't be copied. Third, the promoters and ribosome binding sites you tuned in E. coli may simply not work. Every one of those is organism-specific.

[THEO] So it's like showing up to a foreign country with a phrasebook that's for the wrong language. The words are fine, nobody understands you.

[DR. MARA] That's fair. And for a long time the response was, don't bother — just port your pathway into E. coli. Which works, until it doesn't, because the native host had some capability you couldn't transplant.

[THEO] Which brings us to our oldest paper — Fatma and colleagues, 2020. This is really the manifesto, right?

[DR. MARA] It's a review, and it lays out the thesis cleanly: non-model microbes can serve as cell factories — living production systems for chemicals, fuels, materials — but only if you build organism-specific genetic tools first. And their key argument is that CRISPR changed the economics of that.

[THEO] Because before CRISPR, building tools for a new organism was this bespoke, years-long slog.

[DR. MARA] Right. CRISPR gave you a programmable way to cut or, importantly, to regulate genes using a guide RNA you can just retype. Suddenly the cost of domesticating a new organism dropped. Fatma's review spans Actinobacteria, cyanobacteria, yeasts, fungi — and the through-line is: the chassis isn't the limitation anymore, the tooling is, and here's how CRISPR closes that gap.

[THEO] So that sets the agenda. Everything after this is the field actually doing it. And the first thing you need, if you're building tools for "any organism," is a cloning system that isn't locked to one host.

[DR. MARA] Which is de Vries and colleagues, 2024 — the In- and Out-Cloning work. This is deep in the plumbing, but it matters. They're working in the MoClo world — Modular Cloning, a Golden Gate assembly standard where you snap DNA parts together like defined bricks.

[THEO] Golden Gate — that's the trick where an enzyme cuts outside its own recognition site, so you can design the sticky ends to be whatever you want.

[DR. MARA] Exactly. These are Type IIS enzymes. And the sticky ends — the little single-stranded overhangs — are how parts find their neighbors. The problem is those junctions leave a "scar," a few extra bases between parts. Inside a protein-coding sequence, a scar can wreck your reading frame.

[THEO] So what did they do about it?

[DR. MARA] They used SapI, which leaves a three-nucleotide overhang instead of four. And three nucleotides is a codon. So the junction can be a real amino acid instead of junk — scarless transcription units. Then the clever part is Out-Cloning: instead of hand-building a destination plasmid for each new organism, you assemble it on demand from modular parts. Need an origin and a marker that work in your weird bacterium? Snap them together.

[THEO] That's the phrasebook printer. You don't carry every language — you print the phrasebook for whatever country you just landed in.

[DR. MARA] That's a good way to put it. It directly serves Fatma's thesis: make the tooling generic so a new organism isn't a five-year project.

[THEO] Okay, so now we've got the cloning generalized. Next turning point — actually deploying CRISPR in a real, messy environment. The P. alloputida KT2440 paper.

[DR. MARA] Roghair Stroud and colleagues, also 2024. Pseudomonas alloputida KT2440 is a soil bacterium, a rhizosphere organism — it lives around plant roots. They built a CRISPR interference system, CRISPRi.

[THEO] CRISPRi being the flavor where you don't cut — you use a dead Cas9 to just sit on a gene and block transcription. A dimmer switch, not scissors.

[DR. MARA] Precisely. dCas9, catalytically dead. And they did two things I'd flag. They put it under an inducible promoter — XylS/Pm system, so you control when it turns on — and they integrated it into the chromosome using mini-Tn7, a transposon tool that drops your cargo into a defined site. So it's stable, single-copy, not floating on a plasmid.

[THEO] And the headline?

[DR. MARA] First in-situ gene repression in the rhizosphere — knocking gene expression down while the bacterium is living on actual roots, not in a flask. And they used it to disable pyoverdine, a siderophore.

[THEO] Siderophore — the molecule bacteria pump out to grab iron.

[DR. MARA] Iron is scarce and locked up, so bacteria secrete these chelators to scavenge it, and whoever grabs the iron outcompetes the neighbors. By shutting off pyoverdine with CRISPRi, they could dial that competitive weapon down, in place. That's control of microbial competition in a native setting.

[THEO] So the arc so far: manifesto, then generic cloning, then CRISPR working in the wild. But the last few papers widen the lens — they're not just about tools, they're about which organisms.

[DR. MARA] Right, and here's where it gets interesting. The polyextremophile framework paper argues you can rationally design organisms that survive multiple extremes — cold, salt, radiation — by tracking four root-cause stresses: reactive oxygen species, covalent damage, protein unfolding, and membrane fluidity. Fix the proximal mechanism, predict whether two extremes are synergistic or fight each other.

[THEO] And then nature shows off. The Colwellia piece —

[DR. MARA] Jody Deming's News and Views. Colwellia are psychrophiles — cold-lovers — in sea ice, and the evidence is they operate below the temperature and metabolic limits we predicted. The organism is out ahead of our theory.

[THEO] And the Synechocystis one is the payoff of that whole "worth meeting" idea. They evolved a cyanobacterium to tolerate salt —

[DR. MARA] Adaptive laboratory evolution — you apply selective pressure over generations and let mutations accumulate. They pushed Synechocystis to 6.5% NaCl, and a membrane protein, slr1753, ends up sequestering sodium on the cell surface. They used it toward seawater desalination and reclaiming saline soil — reportedly boosting plant germination substantially.

[THEO] So the field went from "here's why we should bother" to "here's a bug remediating salty dirt." That's the whole arc in five years.

[DR. MARA] And the honest edge is that the tools and the organisms are still racing each other. Colwellia is a reminder these creatures can do things we can't yet explain, let alone engineer.

[THEO] Creatures worth meeting — and apparently, worth catching up to. That's the arc. More after the break.