Ireland’s First Biohackerspace: In Cork

Let me share some extraordinary news with you. After years of watching the biohacker movement rise to prominence around the world, my home city of Cork is suddenly and rapidly becoming a hub of Synthetic Biology, and as part of this transition will soon have a biohackerspace modelled upon the success of La Paillasse, Paris.

In case you don’t want to read more, here’s all you really need to know: there’s an assortment of people and supports nucleating rapidly around a biohackerspace in Cork City, very possibly in a unique and curiously appropriate location. You should be a part of it. Join the DIYbio-Ireland mailing list, if you want to be ready to help build-out the lab ASAP when a location is finalised.

Let me tell you about how this came about.

Firstly; much deserves to be written here about my experience with IndieBB (which, if you are not keeping track, failed to achieve its target funding goal by ~25%), but I’ve got more important things to share, and IndieBB’s backers have already received some updates since the campaign closed. Anyone who hasn’t gotten the news by now doesn’t need it.

So, the story. For months now, an effort has been underway to attract bright ideas and proposals for synthetic biology to an unprecedented startup accelerator program, Synbio-Axlr8r, which will be running over this Summer in Cork. As part of the push to get people to consider Cork as a destination for research and development, we arranged a conference, named The Synthetic Biology Future. The conference would bring local researchers and their overseas colleagues together to discuss the future of synbio and their roles in it, as well as the potential for locations such as Cork as hubs of innovation.

I was prepared for an interesting conference; I’d seen the names on the sheets and been CC’d on some interesting email exchanges. I knew I’d meet researchers and synbio developers from around the world whose work I’d been exposed to previously, and I was looking forward to a great conference.

Several things went unexpectedly well at once, which together felt, to me, (by the day’s end) like an ignition.

A large number of students and postgraduates turned up for the event; the former in part because of strong encouragement from course coordinators, the latter due to a few convincing characters in the local scene getting the word out and bullying people to attend in person.

Then, the delegates started to throw around the word “biohacker” with unexpected comfort. This was prior to any of the “usual suspects” like Synbiota or Thomas Landrain or myself; this was people from without the biohacker community discussing it as if it’s a legitimised part of the synbio movement. It is, of course, but this is the first time I’ve seen non-biohackers say so first.

To reinforce the early, welcoming mention and discussion of biohacking, the lineup already included a number of self-identified biohackers; Thomas Landrain of La Paillasse, Synbiota cofounder Justin Pahara, conference organiser and serial do-er Jacob Shiach, and (humbly) myself. By the time we had a chance to present our talks and our work, the audience had already heard panels discussing the bright future of small-synbio and biohacking, as well as having been regaled by Seán O’Sullivan of SOS Ventures about the importance of risk and “near death experience”. Our reception was, mildly put, warm.

Constant mention was made of the various state and city level constructs that are on the way besides Synbio Axlr8r itself; business parks, earmarked funding, research groups. Absent, I suppose, was my tier; self-driven, open ended research as enabled by biohackerspaces. But, more on this below of course.

Finally, a tidbit revealed early in the day that probably made all the difference was that Cork would be fielding an iGEM team. This came as part of a Cork researcher’s presenting work with synthetic “protein origami”, which was (to me) exciting enough on its own.

At the mixer afterwards (I arrived late after helping with some technical issues in the main auditorium), Bill Liao, whose project the accelerator and conference had been in the first place, expressed an interest in seeing a La Paillasse in Cork, and I have to say it was the first time I didn’t immediately doubt such a thing could happen.

All of this was pretty exciting; we all left the conference feeling uplifted and motivated, I think. I met a lot of great people, many of them in Cork and with a new or revived fascination with the field of synbio, and a taste of the freedom provided by the biohacker mentality.

The next morning, after a late night (Hi Jacob, Thomas, Synbiota & Cork iGEM!) and a failed crowdfunding campaign, I was woken by a call from co-organiser and excellent person Wayne, who told me to be ready to visit a site by early afternoon. There was interest, after the conference; while I was sleeping in (typical) people were planning a biohackerspace, and a potential site was already suggested. Thomas, Jacob and I received a tour of the space, which is amazing and very appropriate, given its history. No early reveals here until it’s final, but I’m excited about this location already.

Over the last few days, the project has gained momentum; plans, communications and collaborations are being put together. A few serendipitous connections presented themselves in that all-too-weird way that they can do. The Cork Biohackerspace is going to happen; it has too much inertia now not to.

This is, to me, the most exciting thing to hit Cork, ever. Obviously I’m biased! But, I’ve been working in my own lab for years, lamenting the company of like-minded biohackers. And, this week I have discovered that there are indeed a sizeable number of like-minded hackers, along with sympathetic academic colleagues, eager supporters, and a network of potential funders and assistants, in my own City. In scant months, Cork will host teams from around the world who are planning ambitious synthetic biology projects, and will incubate and send forth an iGEM team to compete in the jamborees.

And now, we’ll have a place for us all to call home; somewhere to hang our micropipettes and incubate our projects. Somewhere we can apply for a collective license (the EPA explicitly suggested a club to me to simplify licensing, when I applied years ago) and run teaching classes, workshops, tutorials, demonstrations and have fun!

If this excites you as much as it excites me, get thee to the DIYbio-Ireland mailing list, and be ready to help build out the lab. The call could be coming sooner than you think!

IndieBB – Technical FAQ

As I am redesigning the main pitch-page for IndieBB in order to appeal more to the currently-untechnical audience, what little technical detail I’ve reserved for the main page will have to be stripped out; I’m taking this opportunity to write up a “Frequently Asked Questions” post to address the more technical queries I’m seeing in the survey, tweets, comments and other correspondence.

I will soon also post a less technical FAQ, but given that “Less Technical FAQ” is now the design goal for the main IndieBB crowdfunding page, I’m considering it a lower priority.

I can edit and add to this as newer technical questions emerge; please make such queries on the IndieGoGo “comments” pane if possible so other users can see the question and the response!

To the really dedicated techies, I’ll try to return to this with my citations and literature where useful.

Q: What does IndieBB Stand For?

“Indie Biotech Backbone”. Backbone is used interchangeably with “vector” as a term for a plasmid that’s been designed for industrial or research use. Because this is dedicated to Indie Biotechnologists, it’s named appropriately.

Q: Technically Speaking, What’s In IndieBB?

The pitch for “beginners” is that IndieBB makes bacteria fluorescent, but there’s a lot more going on under the hood. To make IndieBB as easy as possible, it includes a novel self-selection system that (if it works as intended) removes the need to include or require antibiotics in the kit. Because this is the most novel element of IndieBB, it’s described further below.

IndieBB will also include a gene for a fluorescent protein called “Green Fluorescent Protein”. For beginners, this is the part they’ll actually notice; from a technical standpoint, it’s actually the least interesting part! Because IndieBB must be a Free-as-in-freedom product, it cannot consist of patented parts. Sadly, in this degenerate age, patents on substantially natural genes are permissible and long outlast their novelty or commercial use, so even very old variants of GFP like the original “enhanced GFP” are still patented. GFP itself was patented, insultingly enough, despite being entirely natural, but fortunately the patent has since lapsed and it can be used as-is.

One noteworthy consequence of sticking to patent-free “natural” GFP is that it comes with all the drawbacks of that protein, now alien to the coddled emerging generation of synthetic biologists. I’ve accounted for the worst of these, the low fluorescence, by using codon optimisation software of my own design to make sure that plenty of the GFP is produced. But one significant drawback remains; it can only fold at ~30C, so cells can’t be cultured at the more usual 37C.

Finally, IndieBB will contain a multiple-cloning site and an origin of replication. Neither are yet set in stone, but I’d like the MCS to be more generally useful than simply for routine cloning; it’ll be biobrick compatible, for starters, and I’d like to offer defined, easily-primed regions (as in, PCR primer landing sites) for use in techniques like gibson assembly; not essential, but maybe useful for some.

At the suggestion of an early supporter, I will also aim to include comparably easy-to-prime sites surrounding the submodules of the plasmid.

Q: Tell Me More About The Self-Selection System

Selection is the process of removing E.coli from the mix who have not absorbed the DNA you offered them, and the ongoing process of preventing cells from losing the DNA afterwards. It is normally accomplished by providing DNA that makes cells immune to Antibiotic, and then applying antibiotics to the culture so that only cells with the DNA survive.

As getting antibiotics for selection is otherwise a large stepping stone for beginners and amateurs to overcome, I’m hoping sidestepping it will facilitate more people self-educating in biotechnology.

Specifically, the system to be used is based on the “Colicin V” (AKA “Microcin V”) system found naturally on some E.coli plasmids in the wild.
Colicin V is a tiny protein that is toxic to E.coli (and only E.coli), but cells that produce the toxin are themselves immune to its effects.
This means that, by copying the natural state of Colicin V, we can use it instead of antibiotics as a self-powered selection system.

Q: Will this (antibiotic-free self-selection) work?

Nothing can be guaranteed. However, I feel confident it will, because it works in its natural context, and behaves predictably in the many in-vitro studies of the system (Colicin V) that have been conducted.

It’s true that the Colicin V system has never been deliberately used as a self-selection system (to my knowledge); if it had, then IndieBB wouldn’t be as novel but at least I could offer some assurance of success. If it works, it’s actually publication-worthy (open access only, don’t worry) in a methods/protocols journal, I think.

Q: Why are you asking for funding for 3 prototypes?

Synthetic Biology is very error prone! In my experience, you need to behave like a Scientist for your first prototype, wherein you learn about the modules and how they behave separately. Once you understand them a bit better, you can start to think like an Engineer and tweak and recombine the modules to achieve the final result.

Specifically, the Colicin V system is best studied first as the “natural” sequence, and I plan to do so using brightly fluorescent, but not open-source (:() proteins so I can better understand it. Once Colicin V has been tested in its “natural” context, and I can say whether it’ll work as-is or need “enhancement”, the second prototype is the same, with an optimised, further modularised, or otherwise altered Colicin V gene cluster, still using enhanced marker proteins to assist in testing.
The final prototype is to be the final product; the optimised Colicin V system plus a patent-free fluorescent gene (optimised wild-type GFP is the current plan) which allows it to be considered “Free/Libre“.

Q: Why are you paying yourself €3000 for 3 months’ work?

It’s unfortunate that this was even asked.

One hopes, firstly, that the questioner is aware that this is far below minimum wage in Ireland. It may come as a surprise, and I am aware I appear much younger than I really am, but I have a family to help support! I might survive on less per month if I lived with my parents and ate noodles all the time, but I have a mortgage to pay for, kids to feed and clothe, and must still contribute to our costs.

For all this, I’m passionate about synthetic biology, and moreso I’m passionate about helping to create an open-source revolution in biotechnology. Sadly, nobody out there is yet offering careers in open source biotechnology, and so I have no recourse but to do it by myself as a full-time job.

€1000/month means that I can justify giving this my full-time work to make it happen. Without that money, I literally would run out of money and would have to seek alternative employment, relegating IndieBB to a weekend project, spared only the time between parenting, work and the necessities of continued survival.

I can guarantee the questioner that any project of comparable complexity that doesn’t include “living wage” in the cost plan is just hiding it among the other costs. One cannot survive upon goodwill alone.

Q: How Will You Handle “Open Source” Development?

At this time, I’m planning to maintain an open repository on a platform that is most suited to DNA. It will be open to access, copying, editing etc., and I will welcome suggestions on the design. However, I’ll be the BDFL on this project, and I don’t promise to accept suggestions if they’re uneconomical, feature-bloating or I just don’t like them. You’re always welcome to synthesise your own projects!

Q: How Will You Handle Open Source Licensing

This is very different, and non-trivial, compared to software, which can be covered by Copyright and those rights then inverted to provide irrevocable freedoms to users. There are strong parallels between the way that code, when compiled, can inherit the copyrights of its source-code, and how one might likewise allow DNA to inherit the copyrights of its “source”. And yet, this is not established or accepted as the norm anywhere that I’m aware of.

I have no intention of patenting the code to apply an irrevocable license, because I don’t honestly believe patents can be used for good.

The Biobrick Public Agreement is a tempting option, but on its own it’s more like the original BSD. It places (very minor) restrictions on how you package materials, and it eschews copyleft. I’m considering making minor alterations to the BPA to suit my needs, but it’s possible I’ll just use the BPA as-is, given my uncertainty as to how to enforce “Free/Libre” in DNA, anyway.

It might amuse readers with a Free/Libre background to know that I did ask Richard Stallman of the GNU Project and Free Software Foundation to offer some advice on the subject, but didn’t get far!

What Will Your IndieBB Be?

IndieBB stands for “Indie Biotech Backbone”. When I started this blog, I had intended “Indie Biotech” to be a phrase that could be generally used, not a “trademark” for my own work, yet people sometimes still refer to my project/company as “Indie Biotech” (I do have a company for this, and it’s called “Glowbiotics”, not “Indie Biotech”!).

With the same spirit in mind, I named my plasmid backbone “Indie Biotech Backbone” because I wanted it to be something that could be used by “indie” genetic engineers worldwide to make their own stuff.

Of course, when trying to pitch for crowdfunding, I’m trying to keep things as broadly-appealing as I can, because I want people to consider trying biotech without feeling intimidated by the more advanced stuff; IndieBB is marketed as an educational system only, when really it’s both educational and developmental.

By this I mean that you can and should use IndieBB to make or discover other cool things. That’s what Free/Libre Biotech means, and it’s what I desperately want to see more of in the world. Some get this right away, but as with any new technology the question arises: What can I do with this?

First, let me answer the unspoken but important “What does the kit teach you to do?”;

 A Beginner’s Guide to IndieBB

IndieBB is a plasmid, meaning a small, circular molecule of DNA carrying non-essential genetic information that does something in (in this case) E.coli.

The IndieBB kit will come with E.coli cells to work with, the IndieBB DNA in a tiny bit of pH balanced water, and (in the full kit) some growth broth, agar, and chemicals used to get the DNA into the E.coli.

The beginner’s guide will include instructions for how to use IndieBB to make the E.coli become fluorescent, but the gist of the experiment looks like this:

  1. Fill out the kit by getting a few ingredients from your local supermarket and pharmacy; rubbing alcohol (often sold at an ideal 70% concentration), powder-free gloves, sterile droppers, cotton wool, and oven bags (or other heat-proof non-metal).
  2. Sterilise jars and the glass petri-dish (assuming you don’t buy pre-sterilised plastic petri dishes) by wrapping in aluminium foil and placing in a cold oven; bring the oven to 200C (fan assisted, please) and leave for 1:30 hours. Do not open the oven during this time, or until the oven has cooled to “warm” or below afterwards; rapid temperature changes can stress or shatter glass!
  3. Make up a small amount of broth by mixing the powder with hot water in clean, sterile glassware, for example jars. Divide it in three and add agar to one jar, and the chemicals used for engineering to another. Then cover the glassware with heat-proof plastic, poke a few holes in the middle of the plastic and lightly tape cotton wool over the holes. This will let air and steam escape when you sterilise it in the microwave, without letting contamination back in (much).
  4. Microwave for several minutes; there is research showing that, for small amounts of liquid, microwaves can be as effective as lab autoclaves at sterilising samples. Rotating plate microwave strongly advised! Expect lots of bubbling and maybe mild overflow, particularly from the agar sample. Once finished, leave sit in microwave for several minutes to avoid risk of flash-boil or burns.
  5. While the microwaving is going on, find a clean room to work in, sterilise a surface using a spray of 70% rubbing alcohol, and arrange your sterilised glass (still in foil) and other ingredients here. If you want to take on a fire hazard, you can set up a vertical blue flame (poor man’s bunsen burner) using a clamp and a blowtorch, but if the air is clear in the room you’ll probably be ok.
  6. When the agar is cool enough to hold but still molten, bring it to the clean area, and pour it into the sterilised petri dish carefully, neither deep enough to overflow nor shallow enough that it’ll dry out quickly and crack. Do this with the lid held a little aside and close the lid immediately to prevent contaminants landing on the agar pool from the air.
  7. While the agar sets, it’s time to grow your E.coli. Necessarily this means that the “flow” of the kit will be interrupted while you await bacteria growth; sorry, nothing that can be done about this. Taking your unaltered broth sample and a sterile dropper, transfer a few drops of the broth into the agar-filled E.coli tube, close the cap and shake, then open and transfer the drops back into the broth sample; this will carry plenty of E.coli back to the tube where they’ll begin to grow and multiply if you leave them somewhere warm. Remember to keep the lid on the broth before and after; it should be open to the air (which should be clean anyway) for as little time as possible.
  8. Waiting for six hours is probably reasonable, but the impatient could probably get away with four. Coming back to your experiment, simply tip your sample of growing E.coli into the jar of modification-chemicals-laced broth, and use another sterile dropper to add a drop or two of IndieBB DNA sample. Close the jar and swirl lightly to mix.
  9. Now leave your cells to transform and to grow. Two things happen here; leaving them for some time in these conditions with DNA leads to some of the E.coli cells absorbing and using the DNA, which means they’ll then start fluorescing and producing a protein (called ‘Colicin V’) that kills the other E.coli not possessing the DNA. After some time incubating, a time-frame not yet known until IndieBB is created and tested, you should have a jar full of fluorescent E.coli.
  10. After this waiting period, you can spread your E.coli on the agar plate you prepared. Nichrome-wire twisted into a small loop around a pen-nib and sterilised by heating in a blue flame until red is best, but using the nib-end of a sterile dropper is probably easier to prepare on short notice. Take just one small droplet of the E.coli from the broth jar using a new, sterile dropper, and eject it onto one side of the agar. Run the nib of the dropper through this little droplet to spread it around one corner of the agar plate, then streak a single line of the spread droplet over into another segment of the agar, and spread that tiny bit around over there. Then streak a tiny bit out of that tiny bit, and spread it around in another segment of the agar..and so on, until you’ve got three or four zones of progressively more spread-out E.coli-plus-broth. We do this because, not knowing precisely how many E.coli are in the tiny droplet, we want to ensure we get one area of the agar where the cells were spread out enough to form distinct little colonies and not just a streak of blobbish mixed cells.
  11. Let the liquid soak into the agar and the surface dry out, then flip the petri dish over so the agar surface is pointing down, and put it somewhere warm to incubate overnight.
  12. The next day, or perhaps the day after that, you should start seeing streaky growth in the first zone of your agar, and then later pinprick colonies form further away, which will grow into circular blobs. Under a UV blacklight (probably not a UV-LED pen-torch, sorry!), hopefully these will be distinctly green! If so, well done! You’ve engineered your first cells.

So, what have you learned?

  • How to sterilise things, how to keep them sterile
  • How to handle and grow bacteria
  • How to get new DNA into bacteria
  • How to identify modified bacteria by fluorescence

Skills you’ll still need to go further, but which the beginners’ guide will offer information on:

  • How genes work in bacteria
  • How genes work elsewhere, by reference to bacteria
  • How to redesign a gene from another species so it’ll work in E.coli
  • How to use software to facilitate this, and how to order DNA

If you can get the hang of the above, as I’ve observed many untrained individuals do since I started following DIYbio, you can start to hack your own project together, perhaps using IndieBB as a ‘backbone’ as it is intended!

Projects Done By DIYbioers

So, what have others in your position done, from which you can draw inspiration? I recommend browsing through the igem back-catalogue for a look at what undergraduate biologists have accomplished, but let’s stick in this post to only those working in a DIY context. I’ll keep it recent, too; I have three nice examples in mind, all of which involve using plasmids and engineering bacteria at stages.

Making Fluorescent Plants for Fun

One is by IndieBB supporter Andreas Sturm, who made a partially fluorescent plant as a project for Ars Electronica. To accomplish this, he used a technique called “agroinfiltration”, where you create a plasmid containing DNA that makes plants do something interesting, and then provide the plasmid to a bacterium called Agrobacterium tumefasciens (you can actually do this using many other bacteria, but A.tumefasciens is the norm). As it happens, A.tumefasciens is one of nature’s more talented genetic engineers, and specialises in injecting DNA into plants; by dipping plants into a broth of plasmid-bearing A.tumefasciens you get a result like Andreas did; a spotty-fluorescent plant.

This is a big deal. Many of the world-changing genetically-engineered crops that are helping to minimise insecticide and atrazine use were modified using the plamid->Agrobacterium->agroinfiltration route, followed by extracting tissue from the modified portions and growing them back into full plants. Andreas was doing this for fun, but if he’d chosen a trait like the B.thuringiensis Cry toxin instead of GFP, he’d have an insect-resistant plant that required fewer or no insecticides.

Andreas is actually involved in a more ambitious, if less practical, project; the famous “Glowing Plant” project that raised absurd funding on Kickstarter months back. The goal is to engineer an open-source easily-grown plant to become fully bioluminescent, so the plants emit a dim glow in the dark! I’ll confess I’m a little skeptical on raw energetics grounds, but it’s ambitious and cool, so I still hope it works for them.

Ag-Hacking with Oxygen-tolerant Nitrogenases (Whew)

Another project that caught my eye lately is Cory Tobin’s research into an obscure but fascinating species called Streptomyces thermoautotrophicus. I’ll confess to you, dear reader, that I (and others) was very skeptical when I first caught wind of Cory trying to isolate a nitrogen-fixing system that could survive oxygen. Did that go over your head? Let me explain. This is a big deal.

Nitrogen fixation” is the process by which bacteria in the soil convert atmospheric gas-nitrogen into compounds of nitrogen that they can use. This process is extremely energy-intensive, and because Nitrogen is so reactive, it’s generally considered something that can only be done under conditions where Oxygen (one of the most reactive elements ever) is not present. Several groups of species that can fix nitrogen (using a system called “nitrogenase“) are well known, and all of them require anoxic conditions to fix nitrogen.

This is important because Nitrogen is one of the critical elements that all life on Earth depends on, and which cannot be used directly from gaseous nitrogen in the air. A lot of the nitrogen we do use gets excreted in forms that break down into gas-nitrogen, becoming lost to the ecosystem. These bacteria are one of the only mechanisms by which nitrogen can be brought back down to the ecosystem-level, and they’re critical for natural habitats as well as agriculture.

What Cory is studying, then, would seem like a White Whale: when he says “nitrogenase which is oxygen tolerant” I hear “impossible”. However, reading through his breakdown of the work so far, it sounds like he really is onto something; a Streptomyces bacterium that can apparently fix atmospheric nitrogen even in the presence of oxygen, a hypothesis he and his collaborators claim they’ve tested by flooding the bacteria with oxygen and a gas-nitrogen isotope. Apparently, they found that the isotope was absorbed by the bacteria, indicating fixation in the presence of oxygen.

OK, I’ll try not to freak out. What’s this got to do with IndieBB? Imagine this is your project; at some point, you’ll want to identify the gene system responsible for this amazing, and potentially world-changing, discovery. If you could get that gene system to work in plants, you could have nitrogen-fixing crops and require little extra fertiliser to grow comparable yields. This is Cory’s goal.

To get there, you’d sequence the genome of your bacterium (done, apparently) and you’d use computational methods to identify genes that are either unfamiliar, or familiar in different contexts which might be involved in this hypothetical new system. You’d then extract each gene system and study it in a more pliable host bacterium; E.coli.

Most interesting gene systems I’ve seen are studied not in their original strain of bacteria, but are instead put into E.coli and studied there. There’s a reason for this; E.coli is so well understood by now that you can say with certainty what is and is not going on, and therefore you can isolate the effects of the added DNA from the context. To be sure your oxygen-tolerant nitrogenase is what you think it is, you must first put it into another context and see if it still works. If it does, then you can think about more ambitious stuff, like using Andreas’ methods to make a nitrogen-fixing plant.

Both steps-study and injection into plants-generally make use of Plasmids to carry the DNA into bacteria and then on into plants. And if you had IndieBB to hand, you could use IndieBB as the backbone of this stage of your project, using IndieBB’s multiple-hacking-site to contain the test-DNA, and using the sequencing primer-sites that will flank the MHS as your sequencing start-points when you want to verify sequence. To get A.tumefasciens to adopt and deliver IndieBB would require a little extra hacking, but honestly I expect someone (Andreas, most likely) will have made such modifications within the year!

There’s two excellent biohacking projects that use plasmids already; one fun and impressive, another exciting and speculative. I hope that’s given you an idea of the scope enabled by a good foundation, and some idea of what your IndieBB might be.

Codon Optimisation: A Hidden Process Behind IndieBB

I’ve posted twice recently after a prolonged blogging absence, and here I am again. Perhaps I should always be running a crowdfunding campaign, so that I have a stake in blogging; I’d be far more prolific! I’m too principled (or, to some “dogmatic”) to include advertisement on this or any blog, and flattr revenue is far too thin to encourage more than the occasional post otherwise.

In any case, today I’d like to share something with you all that I’ve been meaning to write up for ages anyway, but which becomes especially relevant in light of my IndieBB DIYbio/Biohacking/Teaching plasmid design project.

This is the story of how I was faced with a synthetic biology design problem for which there were few acceptable existing solutions, and how it lead me to design my own software for gene design. This software is being used to design IndieBB, so you might have a stake in it already!

Reminder: Like the preceding post, this post gets into the technical details of DNA design. It is not necessary that IndieBB users understand any of this; the kit is designed for total beginners!

Green Fluorescent Protein: Little Green Cells

One of the included genes in IndieBB is the “Green Fluorescent Protein” (GFP) from the jellyfish A.victoria, a widely used biotechnology tool which, when expressed within a cell in sufficient quantity, makes a cell visibly fluorescent. So, colonies of E.coli will be green under a full-spectrum or ultra-violet (UV-A) strip-lamp, allowing you to see you’ve succeeded in making engineered cells.

When the gene for GFP (which, as a protein, had been known of for long before transgenics emerged)green was first discovered, it was a great day for biotech. Finally, a protein that doesn’t need to be extracted from cells to detect; you can just illuminate the cells, and notice that they’re more fluorescent than before! Things got even better when, several years later, an enhanced variant of GFP called “eGFP” was discovered; this was several times more fluorescent and, in modest quantity, was clearly visible by eye. It also had a better excitation spectrum (the range of light frequencies which can make it fluoresce), meaning handheld UV-LEDs could be used instead of UV-striplamps.

(Image credits: Wikipedia)

 

Sadly, while GFP is out of patent by now (it was ludicrous in the first place that a patent could be obtained on a natural gene), eGFP is still burdened by several patents which will not expire for a few years. This means any reasonable hope of making an open source product on eGFP is dead in the water until those patents die, and in the mean-time we’re stuck with GFP.

“Natural” or “wild type” GFP, being significantly less fluorescent than eGFP, needs to be expressed (produced) in cells in much greater quantity, and to achieve this in synthetic biology we do one or both of two things: place the gene under the control of a strong promoter, and codon optimise the gene. The first is a matter of copy and paste, but the latter is far more involved and murky.

Codon Optimisation

If you’re well-read on DNA, you may know that a “gene” is a section of DNA that instructs a cell how to, and under what circumstances to, produce a protein. The protein then goes on to be or do something useful for the cell.

The protein itself is coded for by a sub-set of the gene called the “Coding Sequence”, usually shortened to “CDS”. This happens through a process called “transcription”, where the DNA, starting near the “promoter” region and ending near the “terminator” region is copied to a molecule of RNA. This RNA transcript is then translated according to the RNA code by a protein:RNA complex called a ribosome. Translation uses triplets (codons) of RNA nucleotides to assemble single amino acids into a chain of amino acids, which is called a “protein”.

The ribosome, and the RNA code, are two of the strongest indicators that all known life on earth evolved from a common origin; they are both highly similar across all known species. The RNA code varies between groups of life usually in only one or two places at a time, and even then many of the differences are circumstantial.

However, while the language remains the same, the dialect differs widely. While jellyfish like A.victoria and bacteria like E.coli broadly agree on which three-nucleotide “words” (codons) code for which amino acids, they differ greatly in their relative usage of words which may code for the same amino acid. The RNA code is redundant, meaning that for some amino acids there are many different codons which are used..but some species don’t use some codons at all, whereas other species might use those same codons exclusively. Trying to copy a gene between distantly related species may fail simply because the gene contains “words” which the target species no longer understands, or doesn’t like to use under normal circumstances.

Enter codon optimisation, one of synthetic biology’s darker arts. In principal, if the relative usage of codons can make a protein perform worse in one species than another, then the reverse is also true. Using alternative codons to encode each amino acid of a CDS can make the cell make the protein in higher quantities, or while depleting fewer scarce codons that might be needed for other genes.

As a bonus, while you are re-writing the CDS of a gene anyway, you can specify extraneous things you’d like to do at the same time. Perhaps you want to purge your CDS of target sites for a particular (or many) enzyme, or you’d like to prevent any sequences being created which could form strong secondary structures (areas where DNA folds up into origami) that might interfere with the translation of the RNA code. Let’s call all of this stuff-you-don’t-want “excluded sequences” or “excludes”.

But from that simple principal there is a blossoming of complexity. What’s the best way to optimise genes? At first, people assumed a “best pick” method would work well; analyse the target species, identify the codons that species uses most often, and then always use that codon unless it would create some excluded sequence, in which case you fall back to the second-best, and so on.

Best pick is better than nothing in cases where a gene otherwise won’t work at all. But, the weight of evidence appears to suggest that it doesn’t usually improve gene expression when the gene already works acceptably, on average. Some genes improve, others actually get worse.

An alternative method came into favour which attempted instead to match the relative codon usage of the gene with the relative codon usage of the target cell. So, if upon inspection a species likes to use the four available codons for a particular amino acid in a 4:3:2:1 ratio, then you try to match that ratio in your target gene.

This system actually works better, but it’s still not perfect (though nothing ever will be). But, better refinements weren’t long coming, and there are now several ways of optimising that function, basically, on a random allotment of codons according to a desired final frequency. Some prefer to choose codons according to their relative usage in a subset of cellular proteins believed to be very important, or highly expressed. In at least once case an empirical study determined the best codons to use by mutating many, many copies of some “test” genes and assessing which codons were associated with high expression, though this is expensive and time consuming, and has only been done for E.coli so far.

But at this point, the method remains the same, with the precise details being left to a codon usage table (CUT). Using this method, you specify your gene, and your list of excludes, and your CUT, and your program hopefully churns out a better copy of the gene for the target species, ready to be ordered from a gene synthesis company.

Things seemed fairly stable for a while, until the whole thing got murkier again. First, it became apparent that, once codon scarcity was removed as the primary cause of expression problems, the next most common problem with designed genes was the presence of secondary structures in the CDS region of the gene: the aforementioned “DNA/RNA origami” that can cause RNA templates to fold up, concealing their beginning from eager ribosomes. It would later be demonstrated that, generally speaking, this was most true of the “leader” region where the ribosomes bind and pick up momentum, and less true of the later regions. This is actually (apparently) because the ribosomes themselves, once bound, help to flatten out the RNA and prevent structures from forming.

Once the structure issue was better documented, some bright spark, noticing a few details in “natural” codon usage in wild genes and some contradictory details in custom-made genes, asked whether the speed with which a ribosome translates an RNA template can at times be too fast, the opposite of what everyone else assumed. The assumption made was that, if ribosomes all start at high velocity as soon as they find and stick to an RNA template, then they’ll be thinly spaced as they all progress along the template. If the lead ribosome pauses for some reason; say, it finds a codon that’s used rarely, or the amino acid it needs is suddenly not available when required, then you could get a Ribosome pile-up!

Studies into this actually bore out the idea that ribosomal collisions could occur, and could lead to problems expressing a protein that was, according to the best available knowledge of the day, “highly optimised”. This has become known as the “on-ramp” hypothesis; for good outcomes, the early portion of an RNA template should actually be less optimal than the rest, so that ribosomes start out slow before speeding up later, allowing them to space out slightly and preventing collisions. This appears to be what most wild genes with high transcription rates do, and it appears to be beneficial for designed genes, too.

With me so far? So, the state-of-the-art in gene design today looks like this:

  1. Pick codons according to a frequency table, in preferential order of empirically-determined-to-be-best, used-in-highly-expressed-genes, or overall-frequency-in-the-target-organism’s-genome.
  2. Try to avoid secondary structures in the region that becomes the RNA transcript, particularly in the initial portion.
  3. Try to make the initial portion of the RNA transcript be less optimal than the rest, to create an on-ramp for ribosomes.
  4. Do all of this while avoiding a list of sequences that you’ve determined, for structural, efficiency or convenience reasons, to be better off excluded from the final sequence.

Got that? Now the problem; nothing like this existed.

Codon Optimisation Software Woes

When I began designing genes, I used a tool called jcat, which had a handy web-interface, to design some genes for expression in B.subtilis (readers of prior posts will recall that I was attempting something like IndieBB in that species back then, and that I’ve promised to relate the full story during the crowdfunding campaign).

jcat is a best-pick tool; it prefers to use the most-commonly-used codon for the target species whenever possible. It could exclude sequences from the final design, but seemingly only by specific sequence, rather than using the more useful generic IUPAC-notation (where an expanded set of letters allow you to specify “A or T” or “not G” instead of having to use only A, C, T or G).

I also made use of the gene optimisation system provided as a free service for customers (more on this trend later) by Mr Gene, a since defunct (I think) gene synthesis company, which also appeared to make use of the best-pick system for design. It, too, would allow specific sequences (not IUPAC) to be excluded from the target design, but no IUPAC.

As I became aware that best-pick was being discredited, I started looking around for more up-to-date systems, but I found that all the newer available tools I could find that specified which system they used (best pick or CUT-based) were proprietary, and had very specific terms of use; most were provided by gene synthesis companies who insisted that you buy the resulting gene from them! My more antiauthoritarian users may be like me in balking at this, and may further suggest “screw that, design and buy elsewhere anyway”. But, dear reader, even if I were scurrilous enough to do so (:)), it’s possible that a proprietary program could be embedding a “watermark” in the resulting DNA which is provably linked to the system, and could later result in a burden of intellectual property should the resulting DNA become useful or wildly successful. Also, I’m a purist; why didn’t good software exist for codon optimisation?

So, I wrote my own CUT-based codon optimisation system, and as a bonus I included code that would not only permit exact sequence exclude-lists, but also extended IUPAC-notation exclude-lists. So instead of specifying three separate excludes ["TAG", "CAG", "GAG"], you could just say “BAG”, meaning “[not-A]AG”. I called this system PySplicer (alt Github repo), because it was written in Python (3, obviously) and it eventually made of of a splicing system to rapidly triage a good-ish gene from hundreds of initial candidates, prior to more involved gene editing to resolve excluded sequences.

The first PySplicer was a raw CUT-based design tool with exclude lists, written in the most awful, incomprehensible sort of code. It was one huge script, with one huge Python Class containing tens of methods which each handled a small part of the job, and cross-called one another, sometimes recursively. It was impossible to understand unless you’d written it, and indeed several months later, when I sat down to rewrite the whole thing, I was challenged to understand my own thought process, too! But, at this point I’d fulfilled parts 1 and 4 from the above list of “current best practice in CDS design”.

When I later wanted to rewrite and improve PySplicer, I had by then learned of the on-ramp hypothesis and the importance of DNA/RNA structure to gene expression. I wanted to create the best available gene design software, and that meant implementing all four of the above features. Also, I wanted the code to be not-terrible, so others could see how it worked and help improve it as we learned more about gene design in future. I had been digging into structure analysis tools written for DNA that were written in a now-rare language called FORTRAN, and found them so difficult to read and understand that I felt pity for anyone in my position someday!

Rewriting PySplicer to fill out the list above is a story in computer coding, and I won’t burden the reader, presumably a biology and not programming enthusiast (but possibly both), with the full details.

Suffice to say, fulfilling problem #2, structure analysis, required that PySplicer forfeit being a “pure python” program, because structure analysis is considered one of the harder problems in bioinformatics and requires highly optimised computer code. Not to mention that re-writing it in Python would have required half a year’s work! So instead PySplicer requires that you install a well-regarded and current package written in C called “ViennaRNA“, and then hands off that part of the gene design process to ViennaRNA.

Problem #3 was easier; merely altering the codon usage tables used for the initial portion of the gene suffices to reduce the overall level of optimisation, providing the on-ramp.

Testing PySplicer, Designing a Free/Libre GFP

Of course, programming a codon optimisation tool (by now, it ought to be properly considered a “CDS” optimisation tool I suppose) is meaningless if it’s not used to design genes for testing or use. I follow the philosophy that one should eat one’s own dog-food; I use the tools I propose to others. So, I used PySplicer in a project which involved Green Fluorescent Protein, which I introduced in the above section.

The results were very favourable. While wild-type GFP was often lamented for its poor fluorescence in the available scientific literature, often to the point that it was impossible to see and hard to distinguish, my optimised wild-type GFP was clearly and satisfyingly fluorescent to the naked eye under a small blacklight, even in a brightly lit room and without any filters. This is the green fluorescent protein shown in the crowdfunding video, in fact:

A comparison of E.coli cells bearing PySplicer optimised wtGFP under incandescent and UV illumination. Cells are already somewhat aged, with reduced fluorescence overall and particularly in regions of dense growth.

A comparison of E.coli cells bearing PySplicer optimised wtGFP under incandescent and UV illumination. Cells are already somewhat aged, with reduced fluorescence overall and particularly in regions of dense growth.

So, if you’ve followed me this far, from start to finish, I hope you’ll have a sense of the amount of “invisible” effort that has gone into IndieBB already. In the end of the day, IndieBB (if successful in raising funds) will be a black-box beginner’s project, requiring no knowledge of its working in order for you and others to use it and learn from it. But to provide that level of ease has required a lot of effort, time and experience from me. And along the way, I’ve already been creating and releasing valuable tools for other synthetic biologists (and perhaps you) to use.

A Primer on Plasmid Design (For IndieBB)

So, in the preceding blogpost I introduced my current crowdfunding project, IndieBB:

..If you navigate to the “updates” panel on that crowdfunding page, I’ve been busy keeping things current. Among the promises I’ve made lately was to run through my workflow for total-plasmid-design as I plan to apply to IndieBB.

So, here we go. Before clicking “more”, be aware that you do not need to understand any of the below to complete the IndieBB kit. IndieBB is designed for rank beginners to biotech; this blogpost is intended for ‘advanced’ readers with an interest in my design process.

With that said:

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IndieBB: Open Source Biotech Starter Kit for Everyone

So I started a crowdfunding campaign today, and I’m really happy with the response and enthusiasm so far. I should probably have had a draft blog-post lined up prior to the launch, but my laptop took a dive and I suffered broken-screen-syndrome for 2.5 hours, so things were rushed.

The video in the crowdfunding page on IndieGoGo and embedded here says it all; I want to create a kit that will let everyone get involved in genetic engineering, and from there to help democratise synthetic biology so we can all have a chance to shape the future of technology.

It starts with this; humble, but important. If you agree, please help out by pledging, or by spreading the word!

Reasons to be in Ireland this October: Synbio AXLR8R, Grow Your Own

As is often the case when I fail to post for months, I’ve been busy. In addition to my own work in the lab, which currently entails bypassing and leapfrogging existing affinity chromatography standards while making DIY protein purification trivial (!), I’ve been roped into organising an ambitious Synthetic Biology business accelerator programme in my home city, Cork, and I’m officially a co-curator for the upcoming Synthetic Biology exhibition in Science Gallery, “Grow Your Own“.

Both of these deserve individual attention here. And in both cases it’s worth saying that I’m not the only, or even primary, organiser. Left to my own devices, I’m not likely to instigate a business-focused program, despite my own aspirations to make a living out of biohacking. And were I to do so anyway, it would probably come with a series of stipulations against patents, and perhaps exhorting some manner of copyleft licensing for the outputs. Likewise, while I thoroughly enjoy the sort of exhibit Science Gallery generate, and I have done (and continue to) small work with designers and artists on Synbio projects for exhibits, I’m equally unlikely to personally declare an exhibit and carry it to execution and display.

The reason I’m saying this is simultaneously to avoid stealing undue credit, but also to avoid granting the false impression that, for example, there will be a bias towards “Free/Libre Biotech” in the accelerator, or an undue bias away from centralised-industry in the exhibit. I’ll have ongoing input in both though, so I think/hope both will appeal to those of like mind to myself.

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IndieBiotech.com Migration

Hi all, quick notice: I’m changing my hosting from ixwebhosting.com to 1984hosting.com, because the latter is in a country with a greater regard for freedom of expression.

Some bugs may result! WordPress has a fantastic export/import function that so far appears to work really well, but please let me know if anything’s amiss.

Work at Indie Biotech progresses really well, and I’ll have some exciting stuff to share here soon!

Performing Minipreps with Homebrew Buffers

The alkaline-lysis miniprep is a critical tool in the arsenal of a molecular biologist. It allows one to rapidly isolate only plasmid DNA from a bacterial cell by leveraging the increased resilience of this (usually supercoiled) form of DNA against highly basic conditions.

Minipreps are routinely performed to isolate plasmids that serve as substrates for further assembly work, for PCR amplification of specific gene segments, for direct application in other species or strains, or simply for archival uses (DNA can be easier and cheaper to store than the cells containing it).

However, minipreps are usually done today with kits, using convenient but expensive sets of buffers and binding-columns. Not only are these harder to get as a DIYbiologist, but they actually provide lower yields and may cause shearing or nicking of DNA.

Thankfully, miniprep buffers are not so complicated that they can’t be made at home, provided you can get the required chemicals (some of which are surely replaceable). Preparing buffers takes a while, but you’ll be making volumes of 100mls each; enough for hundreds of minipreps. Here’s how.

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Tuur Van Balen shows how to hack L.delbrueckii on stage

A little while ago, I wrote an article detailing how one might go about hacking S.thermophilus, one of the two strains that co-operate to ferment yogurt from milk.

I chose S.thermophilus randomly of the two; I could as easily have chosen L.delbrueckii. Fortunately, I didn’t, because Tuur Van Balen, syn-bio-artist extraordinaire, has given a practical demonstration for Next Nature on how to do so!

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