koeng

Analog I guess. I'm trying to make a chip for DNA synthesis, and so need physical contact with the real world, with electrodes, where electricity from the circuit will cause localized pH changes, which you can use for precise control of biological reactions. FPGAs can't do that kind of analog work.

Definitely, producing open source works that would help me! Feel free to send me an email and we can talk about what makes sense to you. Also put some more above.

Definitely! If you're willing to help, throw me an email so we can talk where it would best fit for you :)

some sets of problems I have right now (on the hardware / software side):

Hardware: It'd be great to have an open source plate gripper. I wrote a little bit about the general problem of transferring here - https://keonigandall.com/posts/transfer_problem.html . I have a uarm lite6, but wanted to investigate building an arctos robotic arm to move plates between machines. Just need the gripper! This is something I cannot do myself - I have software skills, and can build things with my hands, but have zero skills on designing new hardware.

Software: A lot more here, but depends on interests. I have some general life-improvements I'm looking at doing, but also some wild ideas that need prototyping

In this case, it is literally the Venter genome, just with removed tRNAs / codon sets. I'm collaborating with them on it (they're just doing the final transformation).

good thing we live in real life and not a movie

Someday hopefully soon when I raise a seed :)

I think if you have the right structure, it is easier to train developers to be bioengineers than bioengineers to be developers! Bioengineering tends to be a more wicked discipline, which seriously affects how one writes their code. Makes it kinda crap. Software devs on the other hand typically aren't as experienced in the other field, and so are coming in blind.

yep, nothing in there prevents me from doing work. That article is mainly their legal team fluffing their own feathers for their clients, which I'd hope that one would be able to read between the lines for, considering it was published by the lawyers themselves. The lawsuit itself is quite frivolous and accusing me of stealing and using source code I didn't steal/use, so just have to go through the legal motions to prove that. The more details you know about the case the more absurd it becomes

I'm working on synthesizing a genome at home! Here is a video with more details, as well as a picture of my home lab. I've always wanted to build life from scratch, and I finally have a chance to do it.

Video on what I'm doing - https://www.youtube.com/watch?v=CCiuS1oHnKw

Picture of my home lab - https://x.com/koeng101/status/1844150979484319842

I'm trying to build a DNA assembly company right now (been lots of ups and downs lately...), and one thing I need to do is validate the specs of my oligo pool synthesis provider, Agilent, before I release to customers / raise a seed round. So as a stress-test run of my system, I'm synthesizing a genome, and am thinking about trying to livestream it. The unique technology is variety of ways to assemble and validate DNA from oligo pools for a lot cheaper, pretty much enabling a 10x reduction in DNA synthesis cost vs commercial suppliers. I've worked my ass off for nearly 2 years to get to this moment and am so excited!

The bacteria develop resistance fairly quickly, but you can somewhat get around that with cocktails of phages (like a bunch of phages at once).

> For a sick person after antibiotics therapy and after culture test results, those days/weeks to raise another culture of phages might be deadly

The hope is that you'd have a bunch of phages ready to go from a variety of patients. So you wouldn't need to make a new culture. However, right now they often do personalized phage therapy because they can only get it approved as a last-hope kinda deal. But then it makes the scientists only target chronic infections, because it does take a couple months to get another cocktail of phages ready to go.

> So SQLite in Wasm is just doing compute, and I do all the OS level stuff in Go. No need for Wasm concurrency, cause I can load multiple instances of my Wasm which act like independent OS processes that communicate through the filesystem (SQLite excels at this).

Interesting. So when I am running concurrent readers using your package, it is just loading multiple instances of the wasm code? (I bottleneck to a single writer in the application)

For use in Golang, I really like ncruces wasm SQLite package - https://github.com/ncruces/go-sqlite3 . Unlike cznic's go package (which is great, btw), the wasm version works well on OpenBSD and the like.

They've tried and there are some folks who have made nanopores, but honestly they're SO many years away from Oxford it's almost incomparable.

To just play with a device, I think you can buy the MR1 molecular reality device for like $300, but I don't know that much about it other than it is a solid state nanopore.

Turnaround time is about a day, so as fast as plasmidsaurus would get back to me (I'm also using nanopore, so we're gonna essentially be the same speed, with my library prep taking time but their shipments also taking time). It is mostly cost savings.

I'm working on building a plasmid synthesis company. The price of sequence verified DNA has increased, not decreased, since 2018, even when you account for inflation. On that metric alone, we are regressing in our ability to modify the biological world. I'll be launching in a couple months with a service that costs less than half the incumbent provider, while maintaining their margin. Being able to sequence verify DNA for dirt cheap is a big part of that. Took over a year and a half to get there - it's way harder than it sounds, for a myriad of reasons (things at scale are hard!)

You could mean two things by synthesizing custom RNA - synthesizing oligos (ie, chemical synthesis) or synthesizing mRNA, which is typically from a DNA template (you can have short mRNAs be synthesized as oligos, though). The fundamental problem with oligos is the mutation rates - hence why I sequence verify so much stuff - and you're not really gonna get around that. For other mRNA from a DNA template, well, you need the DNA template in the first place (that's where I come in), and then you usually just transcribe it into RNA using an RNA polymerase. Then, there is essentially an endless rabbit-hole of optimizations for everything under the sun. But fundamentally mRNA only costs the DNA template + polymerase, which you can get for like $75 https://www.neb.com/en-us/products/m0251-t7-rna-polymerase . DNA prices are about $0.09 per base plus $35 right now, I'm aiming for $0.045 per base plus $16.

I do a lot of plasmid sequencing in-house with nanopore, so I have some insights here!

For years, the problem was mainly with the rates of mutations in nanopore sequencing, in particular, non-random mutations. The stuff you couldn't just sort out by sequencing more (well, theoretically at least). This was because of the pore structure. Around 2021, R10 flow cells came out, which were a major improvement on R9 cells. In the protein pore for nanopore sequencing, they basically just stacked two proteins on top of each other, so there are more contact points to get data out of.

With the improvement in nanopore sequencing fundamentals, you now have far fewer non-random mutations. This enables plasmid sequencing. Plasmidsaurus just hit it out the park with convenience of the service.

On cost: most of the cost is in their reliability. Each customer wants their particular plasmid to be actually sequenced - so $15 price tag (and probably much less in-house for them). I currently sequence plasmids for approximately $0.25 in-house, BUT only probabilistically (900/1500 plasmids get sequenced). And to my best approximation, this is a non-random distribution, so just doing it more doesn't fix it. Also you run out of material.

There is an aspect of library prep that is cheaper. Turns out, with illumina sequencing at least, most of the cost is in the indexing step, not the actual sequencing, for plasmids. This is the step of putting barcodes onto the material to figure out which well a particular sequence came from.

Finally, you still do need software! People don't realize how much software you actually have to make to be able to tell plasmid sequence from nanopore sequencing. Plasmidsaurus makes it easy for people. Here is an example from my repo of me figuring out how the hell to interpret some weird data I've gotten:

https://github.com/Koeng101/dnadesign/pull/98

If you want a real example, take a look at this pileup file:

https://github.com/Koeng101/dnadesign/blob/sequencingPileup/...

How to read: https://en.wikipedia.org/wiki/Pileup_format

At position 165, you see a non-random nanopore error that only affects a single side of the DNA, and not the other (there would be lowercase g if the complement was also "mutated"). You need to sort through these to get good data to customers. And that's not as easy (not to mention how often plasmids are mixed!)

It’s really a shame that OpenBSD doesn’t have a good file system. Otherwise I’d use for my production systems (I could put it inside of proxmox, with a ZFS container outside for stability inside, but I like to run bare metal, no VMs)

> can't you just inoculate your starting culture with a larger amount or something

No, I'm doing high throughput cloning in such a way where a limiting factor is going from 1 cell to X cells (usually pickable colonies). So liquid culture doesn't matter quite as much, since I can sequence validate from a colony split (half into colony PCR, half into new culture), and sequencing takes approximately as much time as the growth step takes.

> I'm curious if there is a tradeoff to its super fast growth. Does it have a greater mutation rate?

Eh, not really. The biggest tradeoff is that it isn't studied nearly as much as E.coli, so the chemical competence protocols kind of suck. I can't actually use it in my pipeline right now because of that. So I'm stuck with E.coli until I can figure out a good transformation protocol.

Normal E.coli doubling time, which means about half the speed of Vibrio natriegens. I use NEB turbos sometimes, but they still don't bring down the liquid culture time down from overnight to within a day, mostly.

I would want E.coli to sporulate because then shipping and distribution of strains is a lot easier. This was a problem back when I was shipping at FreeGenes and still is a problem. I used Bacillus subtilis to make the sporenet protocol which worked, but the vector backbone switching was pretty painful.

Clinical strains == not necessarily GRAS. Also, they're clinical, so regulated by MTAs, which are a big no-no.

I could make the E11K purB mutation, but why would I go through the effort in an inferior species of bacteria when I can invest my time into something like Vibrio natriegens? The payoff is better at the end.

I'm expecting that it should grow 2x the speed, sporulate if needed, have natural competence systems for large modifications / genome engineering (lambda red aint there), have some environmental resistances that are uncommon in typical contaminants, have better protein secretion directly out of cloning strain, hell just an expression AND cloning strain, better stability in typical strains that dont require STBLE or whatever, better positive and negative selection markers, Vitamin B1 synthesis pathway for minimal medias, E115K fix in purB for DH5alpha so that they don't grow like crap on M9, the list just goes on.

I use E.coli on a pretty daily basis for cloning. It's alright, and there is so much work that has gone into it as a chassis organism, but overall there are definitely better organisms out there if we just took the time to figure them out (Vibrio natriegens and Bacillus subtilis are two examples).

We absolutely do not have a clear idea of how E.coli works. Hell, we don't even know how almost 1/3 of the genes work on JCVI-Syn3a works, a minimal genome we synthetically created. Far fewer in E.coli.

For synthesis, it’s mainly because there is a monopoly and the challenge (DNA assembly) is super boring in comparison to sexy things like DNA synthesis.

For robotics, it’s because Opentrons started scaling and needed to make more $$$ rather than staying small (fair to them, SoftBank dollars are attractive)

Generally speaking, biology is hard and uncertain, so the market isn’t as competitive as you’d imagine. Synthesis prices stagnated a little bit for a while in comparison to where they could be because Illumina didn’t have competition

Clonal DNA synthesis has increased in price over the past 6 years (even when accounting for inflation). On that metric, we’re actually regressing in our ability to modify the natural world. It’s even worse than stagnation.

Or even look at lab robotics - in 2015, you were able to buy a new opentrons for $2500. Now it’s about $10,000 - the only way to rival the old pricing is to scrounge around used sales.

Enzyme prices haven’t dropped in basically forever. Addgene increased plasmid prices a little bit ago.

I feel like computer hackers can’t even imagine how bad it is over here

For me, this is very true. Most bioinformatics is done in python, but over the last ~4 years I've ported everything I do over to Go: https://github.com/koeng101/dnadesign

I'm just massively more productive, and the fact that I can read code I wrote years ago and fully understand what I was thinking at the time is amazing, and I haven't experienced that in other languages. I've learned other languages quite in depth, but with Go it is simple enough that when I write code, I'm not thinking about code, it is purely the problem being solved, and the code just comes out onto the keyboard.

Ironically enough, I've recently started porting my entirely-go bioinformatics package to be a python package, mainly because I realize I'm not gonna convince everyone else in my field that Go > python

What is the problem with https://github.com/fergusstrange/embedded-postgres/tree/mast... ?

I do exactly the same thing. I’m even running SQLite in wasm so I don’t have any C dependencies. Switching out for Postgres would be awesome

Yeah but then I have to figure out the pocketbase API and keep it as a dependency. I'd rather the dependency footprint be as small as possible, accepting the minor tradeoff of having to implement auth myself.

I have a go monolith, htmx for dynamic pages, and SQLite to back it. I really tried to find something for user login / auth / forgot password, but ended up just implementing it myself. Way easier than figuring out APIs and such to integrate with my pages.

I'll make sure to explain it a little more!

Basically, I developed a whole lot of bebop/poly, but I had some disagreements with the owner of the repo (Timothy Stiles) about direction at the later stages of development. For example, I wanted to standardize all the parsers to use a generic interface, so that they're all used in the same way, while he didn't really want to change anything. There were other features I wanted to add as well - you can see a full list in the changelog (which are the changes since diverging from upstream)

I pretty much rebuilt my bioinformatics stack from scratch in Go, and I’m pretty proud of it! It does things that no other library, even python libraries, do for synthetic biology applications.

I’m pretty sure nobody else uses it, but I use it a lot for DNA design work for my company https://github.com/koeng101/dnadesign

They added the star as an edit because of my playground link. So, neither

That’s not true. Here is a go playground of pretty much that exact code running just fine

https://go.dev/play/p/v7OM9s_pRqY