I worked in Syn Bio from 2008-2010, long before CRISPR came onto the scene. I worked on a couple of iGEM projects, that's the international genetically engineered machines conference at MIT.
The basic premise was we would design E. Coli "strains" or "models" that had foreign DNA inserted via commonly-shared genetic tools. It is comparable to using Linux as a shared OS, lots of other FOSS developers helping each other... except that to get totally new DNA into your E. coli, you had to have it shipped in on dry ice from a willing lab halfway around the world. So, you can see why the field is much smaller and more niche than anything software-related...downloading a library is WAY faster and cheaper than shipping in a new genetic module.
CRISPR would've been a game changer for us. We could've much more easily edited gene sequences, though it still would've been hard to get foreign DNA from other labs...I don't believe CRISPR is good for writing huge new sequences... But small changes, small modifications, would've been MUCH faster with CRISPR. We could've gotten a foreign DNA sequence and then tried out 100 different mutations very simply, perhaps discovering an even better functionality than nature's original code.
Crispr is not necessary for e coli mutagenesis. For plasmids you really want to use Gibson assembly. For chromosomal modification lambda red is as easy as crispr.
Gibson assembly is basically foolproof. I trained an intern with almost no molecular biology wetlab experience and in three months he designed DNA for and made fifty mutants (I picked whuch mutations to try) and ran biochemistry on half of them:
You're right that you don't need CRISPR for genome engineering of S. cerevisiae (baker's yeast) or E. coli.
However, having worked with both prokaryotes and eukaryotes it definitely makes life easier. During my PhD (about metabolic engineering of S. cerevisiae) CRISPR started to get traction. Before I could incorporate one change at a time. Using CRISPR/Cas9 I could do up to 6 (at different places). That's a big deal!
I also PoC'ed CRISPR in a bacterium (C. glutamicum). Also there it opened up new avenues.
I had a guest lecture from a couple of researchers working on bioproduction that mentioned crispr let them speed up their design process for new e coli strains, but yeah in general it's not too difficult to hack together a pathway with a Gibson (unless you want to do more than 5 parts in the assembly which gets finicky). Or a golden gate if you can get the sequences for the overhangs right. But I will say I've worked in a lab with at least one person who wasnt able to perform a relatively simple gibson somehow, so the principle that the universe will always find a greater fool still applies
OK, so gibson assembly is not completely foolproof, when I first started working with it I thought the whole thing was a joke because I spent a month trying to get even one clone, and it didn't take. Turned out the site we were trying to (TetR IIRC, it's been 10 years so I'm not sure) clone into had a strange tandem inversion sequence flanking it. If you stick to cloning into MCSes or assembling sequences you've checked/built yourself it should be fine (the liklihood of problematic random tandem inversions is low).
a 1:1 cloning is very much doable with terrible hands. You can reasonably do up to 8 pieces in the assembly, but as you get beyond 4 pieces it becomes important to follow the instruction exactly.
The craziest thing I did, though, was I cloned a (small) eukaryotic chromosome into an E Coli, by gibsoning against the telomere sequence.
At some point the difference becomes using very specially prepared competent cells and being very careful about how you get them in (the mechanism of electorporation is... not obvious), and of course one of the tricky things about gibson is that it's super salty, meaning that you have to be careful when electroporating.
For doing mutagenesis on plasmids, you don't even need gibson. Overlapped primers do some weird circularization during PCR, so for point mutations you don't even need gibson mix.
I'm personally a big fan of synthesis + GoldenGate for building genetic circuits. Gibson, IMO, destroys reproduciblity (how many different PCR kits are there?) and leaning towards Gibson assembly over enzymatic assembly methods (GoldenGate, maybe even BioBricks) makes biotech an Ivory-Tower exercise, since not everyone has access to oligos.
I don't know about that. I've bought 50-mer oligos at $20 a pop to do a 6-part assembly in my garage (I made a strain of yeast that generates cannabinoids). DNA oligos "basically last forever" if you handle them relatively correctly, even in a terrible defrost-cycling consumer freezer. Enzymes, especially specialty ones, not rock solid ones like modern TAQ, have a very short halflife in your freezer, especially if your freezer is consumer-grade.
> how many different PCR kits are there?
Yeah, just buy NEB. Seriously. Not worth screwing around with companies that have lower QC standards.
NEB doesn’t ship residential, and there are many parts of the world that you can’t buy $20 oligos from. I like doing pretty complicated builds, and so I’d go bankrupt pretty much immediately because I don’t just build one thing.
How is gibson ivory tower? It's literally just based on overlapping sequences. And how is it hard to reproduce? You select out colonies and verify with sanger. These are not things that are inaccessible to most people, especially if you live near a DIY bio hacker space, and if you get involved in the biohacker community you'll get a sense of what providers ship to residential address, where to get certain equiptment, and so on. Just googling will get you information that can help you figure out how to get what you need.
Reordering of oligos is expensive, which makes people not reproduce. Sanger sequencing is pretty expensive ($5 sure, but over 5kb on 3 different constructs becomes pricey). PCR machines also ain’t that cheap (when you’re a noob and don’t know what to buy), especially when you need to do more than 8 PCRs at a time.
In theory, works in practice, and in practice, works well in theory. Even for experienced DIYbio folks, cloning isn’t just 1,2,3.
Also, PCRs themselves aren’t very reproducible. It’s not about the overlapping sequence.
I find synbio terrifying. Think about what you know about the dysfunctions of our field, IT, but with hardware-that-is-software and can eat you or make you sick. A computer virus can infect the Internet, a virus can infect the real world.
We should study life for at least two to five hundred more years before attempting applications via these methods.
Genetic engineering applications (except for human health) should be banned, IMO, until we have enough information to do it responsibly.
We already have species and methods of creating new variations that answer our needs.
> Luther Burbank (March 7, 1849 – April 11, 1926) was an American botanist, horticulturist and pioneer in agricultural science. He developed more than 800 strains and varieties of plants over his 55-year career.
Couple that with the recent fast technological advances (ex: rejuvenating stem cells), the many possible applications, cheap capital, and I see all the ingredients for a boom.
Just like the 2000s were about internet and the 2010s were about fintech and cryptos, the 2020s may be about biohacking
The problem is that the aforementioned advances tend to often be 1) Patent encumbered, 2) Difficult to scale and put into production i.e. works only under very specific conditions in the lab with very exact set of dependencies required. 3) FDA the ultimate gatekeeper. There are multiple ways to improve upon (2) but the first and last can only be changed through legislation and the biotechnology establishment is perfectly happy with the status quo (yay for regulatory capture and rentseeking). Credentialism is also a major problem, good luck raising funding without a graduate degree (or several).
I (do not) look forward to seeing the results of "failing fast" here. The conspiracy theorists are convinced that this is how we got to COVID19 in the first place, and while I'm not really convinced there, I really do not enjoy thinking about the potential of such technology to create bioweapons of a scale never seen before.
SynBio in the 2020s smells a bit like green tech in the 2000s. I see a ton of founders and funders with zero biology background thinking that they have the vision and can just hire biology expertise.
Biology is ruthlessly unforgiving and complicated, and often times very low efficiency. For example, it’s a bit amusing to see the ratio of CRISPR mentions in startups vs the techniques actually being used by scientists. You would never use CRISPR if you had the option of using Cre. I’ve made several stable transgenic animal lines using Tol2 transgenisis, which is much higher efficiency, too.
If you’re excited by the space, but don’t have a biology background, consider joining an organization led by a biologist.
This was my experience working in SynBio -- the iteration cycles are so much longer, the failures so much more complex, that someone who comes from a software-first background has a hard time because it just doesn't scale as nicely.
If we look at the space, I think it is clear that biologist first companies have been the clear winners up to this point -- Gingko Bioworks being one clear example
> I see a ton of founders and funders with zero biology background thinking that they have the vision and can just hire biology expertise.
Genuinely curious about your data here. The two first companies mentioned in the article (Zymergen and Gingko) have founders with biology background. Many other companies I know as well.
> You would never use CRISPR if you had the option of using Cre. I’ve made several stable transgenic animal lines using Tol2 transgenisis, which is much higher efficiency, too.
I invest a little in the space so have seen some pitches at IndieBio, YCombinator, and talked with a bunch of companies. There are indeed some really great founders with biology background as you point out. But have been seeing more and more founders from software background. The enthusiasm is great, and people with software backgrounds have a ton to contribute! Just maybe shouldn’t be the CEO.
> baker's yeast, my preference would definitely be CRISPR/Cas9
I’ve never worked with yeast but was under the impression that you could just give them plasmids and call it a day?
You've also got a huge number of DIY biohackers with no business background or vision for a commercial enterprise hacking away in diy hackerspace around the country. I think those hackers will evolve into the truly successful next generation of synthetic bio companies, like what happened to tech companies.
Or, they’ll follow the path of entrepreneurship from makerspaces in 3D printing and found 2000 nearly identical companies all making an FDM printer. Synthetic biology is fundamentally different from software and much harder to build a business than even hardware. That said, the defensible moat is even stronger and there are indeed huge opportunities!
I'm well aware of how much harder it is in this space haha, I've spent the past couple years building a company in this space and have run into to all the roadblocks. The advantage of the environment now is that large numbers of people can spend years hacking away at some bio experiment or problem they're interested in for an amount of money that's not out of reach for the upper middle class. There's already the equivalent of a flood of 3d printer companies - so many people working on DIY thermocyclers/gelboxes etc, there's a running joke that a lot if people spend more time making tools than using them to actually do biology, but with time you might see people using those tools to build interesting things. It's not as easy as doing electronics in your garage in the 1970s, but the inflation adjusted price point is probably equivalent (for the equiptment that is - the house the garage is attached to probably costs quite a bit more...)
On a tangential note: I've seen the upcoming rise of Syn Bio for quite some time now. But I've never figured out, how to participate / help its growth as a pure SWE.
Job offers related to that field require a skill stack, that I have zero investment in. I'm also in my thirties, so starting from scratch isn't really an option. Someone else here from a similar background, who managed to get a foot into the Syn Bio industry?
edit:
Should have mentioned, I'm currently living in Europe. Although I might give that up in order to break into the industry.
There is a niche of companies that are starting to let software eat biology. It's one of the latecomers to utilize the improvements, but they do exist.
[My company is one - we have pure software engineers on our team, we build synthetic therapeutic proteins]
Check out companies like Zymergen, Benchling, Synthego, Bolt Threads, Gingko, Riffyn, & Teselagen.
In terms of pure software, helping open source scientific libraries, image processing for science, and genomics software would help out what is otherwise an under-funded field.
We're actively hiring SWEs now. Particularly folks with your experience level. The opportunities for software engineering at the intersection of bio, materials and computing are mind-bogglingly challenging, fun and satisfying. Send your inquiries to join@riffyn.com
It mystifies me how often Amyris is omitted from posts about synbio. Sure it's not a hot startup, but it has commercialized more molecules than anybody in the industry (and possibly as many as everybody else combined).
30 comments
[ 2.2 ms ] story [ 71.0 ms ] threadThe basic premise was we would design E. Coli "strains" or "models" that had foreign DNA inserted via commonly-shared genetic tools. It is comparable to using Linux as a shared OS, lots of other FOSS developers helping each other... except that to get totally new DNA into your E. coli, you had to have it shipped in on dry ice from a willing lab halfway around the world. So, you can see why the field is much smaller and more niche than anything software-related...downloading a library is WAY faster and cheaper than shipping in a new genetic module.
CRISPR would've been a game changer for us. We could've much more easily edited gene sequences, though it still would've been hard to get foreign DNA from other labs...I don't believe CRISPR is good for writing huge new sequences... But small changes, small modifications, would've been MUCH faster with CRISPR. We could've gotten a foreign DNA sequence and then tried out 100 different mutations very simply, perhaps discovering an even better functionality than nature's original code.
Gibson assembly is basically foolproof. I trained an intern with almost no molecular biology wetlab experience and in three months he designed DNA for and made fifty mutants (I picked whuch mutations to try) and ran biochemistry on half of them:
https://pubmed.ncbi.nlm.nih.gov/24934472/
For yeast, you need neither. Just drop the DNA in and the yeast takes it up!
However, having worked with both prokaryotes and eukaryotes it definitely makes life easier. During my PhD (about metabolic engineering of S. cerevisiae) CRISPR started to get traction. Before I could incorporate one change at a time. Using CRISPR/Cas9 I could do up to 6 (at different places). That's a big deal!
I also PoC'ed CRISPR in a bacterium (C. glutamicum). Also there it opened up new avenues.
a 1:1 cloning is very much doable with terrible hands. You can reasonably do up to 8 pieces in the assembly, but as you get beyond 4 pieces it becomes important to follow the instruction exactly.
The craziest thing I did, though, was I cloned a (small) eukaryotic chromosome into an E Coli, by gibsoning against the telomere sequence.
At some point the difference becomes using very specially prepared competent cells and being very careful about how you get them in (the mechanism of electorporation is... not obvious), and of course one of the tricky things about gibson is that it's super salty, meaning that you have to be careful when electroporating.
I'm personally a big fan of synthesis + GoldenGate for building genetic circuits. Gibson, IMO, destroys reproduciblity (how many different PCR kits are there?) and leaning towards Gibson assembly over enzymatic assembly methods (GoldenGate, maybe even BioBricks) makes biotech an Ivory-Tower exercise, since not everyone has access to oligos.
I don't know about that. I've bought 50-mer oligos at $20 a pop to do a 6-part assembly in my garage (I made a strain of yeast that generates cannabinoids). DNA oligos "basically last forever" if you handle them relatively correctly, even in a terrible defrost-cycling consumer freezer. Enzymes, especially specialty ones, not rock solid ones like modern TAQ, have a very short halflife in your freezer, especially if your freezer is consumer-grade.
> how many different PCR kits are there?
Yeah, just buy NEB. Seriously. Not worth screwing around with companies that have lower QC standards.
https://medium.com/@ThatMrE/a-guide-to-diybio-updated-2019-a...
In theory, works in practice, and in practice, works well in theory. Even for experienced DIYbio folks, cloning isn’t just 1,2,3.
Also, PCRs themselves aren’t very reproducible. It’s not about the overlapping sequence.
We should study life for at least two to five hundred more years before attempting applications via these methods.
Genetic engineering applications (except for human health) should be banned, IMO, until we have enough information to do it responsibly.
We already have species and methods of creating new variations that answer our needs.
> Luther Burbank (March 7, 1849 – April 11, 1926) was an American botanist, horticulturist and pioneer in agricultural science. He developed more than 800 strains and varieties of plants over his 55-year career.
https://en.wikipedia.org/wiki/Luther_Burbank
Couple that with the recent fast technological advances (ex: rejuvenating stem cells), the many possible applications, cheap capital, and I see all the ingredients for a boom.
Just like the 2000s were about internet and the 2010s were about fintech and cryptos, the 2020s may be about biohacking
Biology is ruthlessly unforgiving and complicated, and often times very low efficiency. For example, it’s a bit amusing to see the ratio of CRISPR mentions in startups vs the techniques actually being used by scientists. You would never use CRISPR if you had the option of using Cre. I’ve made several stable transgenic animal lines using Tol2 transgenisis, which is much higher efficiency, too.
If you’re excited by the space, but don’t have a biology background, consider joining an organization led by a biologist.
If we look at the space, I think it is clear that biologist first companies have been the clear winners up to this point -- Gingko Bioworks being one clear example
Genuinely curious about your data here. The two first companies mentioned in the article (Zymergen and Gingko) have founders with biology background. Many other companies I know as well.
> You would never use CRISPR if you had the option of using Cre. I’ve made several stable transgenic animal lines using Tol2 transgenisis, which is much higher efficiency, too.
That probably depends on the organism you're working with. E.g. with baker's yeast, my preference would definitely be CRISPR/Cas9 instead of cre recombinase (see e.g. https://academic.oup.com/femsyr/article/15/2/fou004/534426 ).
I invest a little in the space so have seen some pitches at IndieBio, YCombinator, and talked with a bunch of companies. There are indeed some really great founders with biology background as you point out. But have been seeing more and more founders from software background. The enthusiasm is great, and people with software backgrounds have a ton to contribute! Just maybe shouldn’t be the CEO.
> baker's yeast, my preference would definitely be CRISPR/Cas9
I’ve never worked with yeast but was under the impression that you could just give them plasmids and call it a day?
Job offers related to that field require a skill stack, that I have zero investment in. I'm also in my thirties, so starting from scratch isn't really an option. Someone else here from a similar background, who managed to get a foot into the Syn Bio industry?
edit: Should have mentioned, I'm currently living in Europe. Although I might give that up in order to break into the industry.
Disclaimer: I'm an investor in TeselaGen.
[My company is one - we have pure software engineers on our team, we build synthetic therapeutic proteins]
Check out companies like Zymergen, Benchling, Synthego, Bolt Threads, Gingko, Riffyn, & Teselagen.
In terms of pure software, helping open source scientific libraries, image processing for science, and genomics software would help out what is otherwise an under-funded field.