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!
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.
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!
An article covering my work was published today in the online edition of MIT Technology Review Magazine. It was written by Antonio Regalado, and he was very thorough in his research; nobody else to date has asked for scanned documentation to back up my claim of winning a Class 1 License from the EPA!
The article was well written and had a great narrative flow, but some readers may be left with questions on the “crunchy bits”: details that don’t make a great story, but might satisfy a sufficiently piqued interest. So, here’s my attempt at an FAQ.
There is a common conceit among we DIYbio enthusiasts, namely to suggest that one could opt to create “glow-in-the-dark yoghurt” using DIYbio-oriented techniques as a nigh trivial matter. Indeed, this conceit led to my recently being queried by twitter and email about the possibility; where are the guides and how-tos, if it is so trivial?
While a conceit it may be to suggest that glow-in-the-dark yoghurt would be trivial, that’s not to say it’s at all out of reach to the dedicated biohacker. Here, I will lay out a suggested course of action based on the available literature.
Firstly, let use choose our definitions. What do we mean by “glow in the dark”? There are two commonly pursued strategies to choose from; fluorescence and bioluminescence. The former means that the bacteria will glow some colour when exposed to blue or ultraviolet light, usually green. The latter means that the bacteria will literally glow, emitting their own light from within provided they have enough energy from food.
Though bioluminescence is without doubt a cooler trait, for a variety of practical reasons, fluorescence is a more practical choice. Until, that is, one has more experience in yoghurt hacking and more money to burn on ambitious goals! So, here we will explore the transformation of a yoghurt bacterium with a variant of “Green Fluorescent Protein“, which renders organisms fluorescent under blue or UV light.
So, now that we know what we want, what are we working with? Yoghurt is most often composed of a co-culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, shortened hereafter to L.bulgaricus (its former name). The two species work in concert, providing nutrients and cofactors to one another and producing acids and bacteriocins that prevent invasion of other unwelcome species. Together they digest and remove much of the lactose content of the milk, and produce the polysaccharides and volatile molecules that give yoghurt its distinctive mouthfeel and flavour.
Of these two, either could be engineered to fluoresce, though the construction of the DNA and the methods needed to get it into the cells would differ. For this example, let us say that we have chosen to engineer S.thermophilus (chosen at random; I haven’t even researched L.bulgaricus, and it may indeed be a far easier bet for this project! Expect an update later if so.).
A quick literature hunt on DuckDuckGo and Google Scholar reveals that the most popular method for engineering S.thermophilus is through induction of a state called “Natural Competence“. In this process, cells are encouraged to activate a natural system of DNA uptake by exposing them to starvation or stressful conditions. This system is composed of multiple regulatory systems, and involves proteins that actively bind and absorb DNA, and proteins that splice this DNA into the cells’ chromosomes if it matches them at least partially. Not all strains respond to the usual methods used to induce competence, and some strains are considered nontransformable even though they are known to contain a seemingly intact competence gene array.
This poses a problem to DIYbioers, who may not know whether their chosen strain responds to induction of competence. However, a deeper search through the literature reveals a more universal method. This method uses an artificial pheromone called ComS(17-24) (AKA Shp316 which consists of the amino acids “PYFAGCL”), which mimics the natural induction pheromone required for competence to develop. Interestingly, even in strains where competence cannot be induced normally, addition of ComS induces competence provided the necessary genes are intact. Therefore, this seems a safer bet for DIYbioers seeking hacked yoghurt!
You might be thinking at this point, isn’t that an awkward thing to get? Firstly, the alternative involves starving the cells in an extremely awkward “chemically defined medium”, the ingredients for which are likely to be expensive if desired in moderate purity. Secondly, the pheromone is a short peptide (a very small protein), and there are several companies offering cheap peptides; $2 per amino acid seems to be the usual price. For the ComS peptide (“PYFAGCL”), that would amount to $14, although expect to pay tax and refrigerated shipping sufficient to raise the price to perhaps $40 (or more?). For that, you will probably get quite a few transformations done!
Methods employed for induction of competence (see methods in this paper for an example) through ComS seem to bear a few artefacts of prior methods, including the use of chemically defined medium and prolonged incubation times with DNA prior to the addition of ComS. The use of Chemically Defined Media was originally necessary to induce competence by starvation, but with ComS added artificially this shouldn’t be needed anymore. Although I have *not* tested either the referenced methods or my suggested updates, here is a protocol that ought to work based on my understanding of the process and the function of ComS:
“Rich Broth” in this case is usually an exceptionally rich broth called “M17″, which includes beef extract, yeast extract, pre-digested protein, glucose, and a few salts and phosphatase inhibitors, sometimes even with lactose added for good measure. This is almost without doubt overkill. A broth consisting of skimmed milk powder and a bit of Yeast Extract (without added salt) will certainly suffice. Let’s say 20g Skimmed Milk Powder and 3g Yeast Extract in 1L deionised water.
You’ll note that in the above protocol, the DNA is required to have 1kb+ complementary ends to chromosomal DNA. That’s because the uptake of DNA is only the first part of the process; the DNA has to integrate into the cells’ own chromosomal DNA in order to be maintained for longer than a generation or two. To achieve this, you have to provide DNA that has flanking complementary regions long enough to encourage a process called “homologous recombination“, wherein the cell exchanges part of its normal DNA for the provided DNA. In order to select for the subset of cells that do exchange their DNA thusly, it is common to use antibiotic resistance genes, so that non-transformed cells can be killed.
Once cells have been selected, antibiotics are probably not necessary in order to ensure the DNA is inherited by daughter cells, as it is part of the chromosome. Since antibiotics are not needed once the gene is established, it would be responsible to remove the cassette afterwards, even if it introduces additional complications. This is too complicated for a “beginner’s guide to hacking yoghurt”, but should be strongly considered; I’d suggest an inducible Cre/LoxP system, and would be happy to consult and assist anyone wishing to attempt this.
For our project then, we want DNA that resembles the following:
Colourised and labelled sections of the DNA, presented with the unnamed section of target DNA. This assumes the simplest scenario where no "cleanup" mechanisms are included to remove the resistance gene.
The promoter (Prom) should be a constantly “on” promoter, termed “constitutive”. The terminator (Term) is a region of DNA that prevents the gene from transcribing beyond its normal context, which could cause unintended interruptions of cellular functions; unhappy bacteria could result, and the gene could end up unstable. The antibiotic should be chosen to avoid medically significant antibiotics such as ampicillin; this is a civic responsibility matter, as otherwise your yoghurt could end up assisting dangerous pathogens in becoming resistant to medicines. Ideally your antibiotics would be self-excising once they become unnecessary, leaving a yoghurt containing only harmless fluorescent proteins and nothing else.
The homologous regions (Flank1 and Flank2) you choose are down to where in the chromosome you wish the DNA to end up. There’s a few million letters of DNA there to play with, and quite a few unnecessary genetic bits you could replace if you chose to. There are probably some retroviral genomes hiding away in the average S.thermophilus genome that you could deliberately replace, for example. However, a design consideration to bear in mind is the presence of native restriction enzymes; enzymes that chop up DNA when they detect certain “words”. In S.thermophilus, there are quite a few such enzymes, and the most common “words” they target are: CCWGG; CCCG; GGCC; CCGG; GATC; GCSGC; GCNGC; CCWGG.
Although native S.thermophilus DNA won’t be cut thusly, due to the protective effects of the same restriction systems, any newly added DNA will be, and the efficiency of transformation will suffer greatly. Therefore, choose target DNA that doesn’t contain these words, or contains as few as possible. Also make certain to screen the newly designed DNA for these words, modifying as necessary to remove them. Most codon adaptation tools will offer to exclude sites, and manual removal from other DNA may be required. If you don’t know what you’re doing manually editing promoters and terminators, opt first to use different promoters/terminators rather than risk failure of the DNA entirely.
Once you’ve identified the target sites, order PCR primers to amplify the two flanking regions using PCR. You’ll need a good enzyme like Pfu or KOD rather than old-school “Taq”, because taq can’t reliably amplify large enough regions. Splash out and treat yourself to a good kit, or homebrew some from a Pfu-producing strain.
Then design and order your DNA: choose a GFP derivative of your liking, there are plenty to choose from. Alter the codons in the protein to minimise those that S.thermophilus doesn’t use, so that it expresses well. Don’t bother with a “best codon” optimisation strategy, as these strategies often fail to achieve the expected results. Just use tools like JCAT (downloaded locally, rather than the online tool) to identify which codons are least desirable.
To do this, find the S.thermophilus genome in NCBI, download it, and feed it into JCAT so that JCAT can build a profile. Then provide the desired GFP derivative to JCAT, and let it calculate which codons are least optimal; replace these so each codon is at least above the 50% margin. JCAT is designed to automagically make all the codons near-perfect, but manual alteration of just the limiting codons is all that’s needed. Sometimes, having all-perfect codons can be counterproductive, for reasons not fully understood yet.
Once you have the protein as optimised DNA, attach a constitutive promoter; you can probably find one by searching the literature, nicking it from a critical metabolic gene, or a virus that predates the species. Also add a shine-dalgarno sequence to your protein coding sequence, just in front of the start codon. If you can’t find an appropriate Shine-Dalgarno, the complementary sequence to the last few nucleotides of the 16S rRNA (changing Us to Ts) in S.thermophilus should be ideal. That is, after all, what Shine-Dalgarno sequences usually seem to entail; target sites for 16S rRNA binding.
Finally, add a terminator. Terminators should be fairly species independent, as their function is down to their RNA structure forming as they are transcribed. Therefore, you can probably use a good, well-tested terminator from the parts registry, even one from E.coli. However, if you can find a nice B.subtilis terminator, stick with that; it’s more closely related to S.thermophilus than E.coli. Best be on the safe side?
The resistance cassette can be built likewise, but it’s easier to just find one of the plasmids commonly used for studying S.thermophilus genetics, of which there are several, and to copy/paste the DNA corresponding to the resistance gene out of one of these. Be careful to ensure that the annotated DNA includes a full gene and not simply the coding sequence for the resistance protein; your resistance gene does need to be transcribed in order for it to work! So, if the “resistance cassette” starts with ATG and ends with TAA, be careful to include a wide margin of DNA around it, perhaps 150bp to either end to be on the safe side. If you actually have a plasmid matching one of these in stock, or can source one, just get it and PCR the DNA out to save money.
Once you have the desired DNA for expression of the GFP protein and the resistance protein, you need to flank it with the intended target sequences. In order to achieve this with the PCR-amplified target sequences, you need to use another round of PCR to assemble the two flanking ends to the custom designed DNA; for this to work, there must be primer-length regions of complementarity between the amplified flanking regions and the designed DNA.
In order words, add some DNA to either end of the insertion cassette that matches at least 20bp of the inner ends of the flanking DNA. Then, order your DNA and wait a month or so. You’ll be paying about 28€c/bp; that’s what I was charged by Epoch, at any rate (whom I can highly recommend).
When the DNA arrives, the procedure goes like this: use PCR to amplify the flanking ends of the target site, and to amplify your intended insertion cassette. Use another round of PCR to fuse the “left” flank, the insertion sequence, and the “right” flank together. Use a third round to amplify from this the finished DNA, by using the upstream (“forward”) primer of the “left” flank and the downstream (“reverse complement”) primer of the “right” flank. This will certainly call for a good enzyme, as you’ll be amplifying in excess of 3kb of DNA.
This PCR product can then be directly added, along with 1uM of ComS (which you should also have received in the post by now), to your washed, diluted overnight cells as described above. The cells should, if all goes well, be induced to active competence, absorb the DNA, and include it in their chromosomes. When you apply antibiotic selection after 5/6 hours of incubation, you’ll kill any cells that *didn’t* undergo this transformation; there may be very few cells indeed that survive due to correct transformation.
With any luck, you’ll see colonies on the agar the next day; try flashing a blue LED at them and see if they fluoresce the desired colour! You can filter out blue using an orange filter, which can be as contrived as a sweet wrapper. If you use UV light, you’ll probably have a clearer view of any fluorescence, because UV doesn’t appear very bright if it’s visible at all.. but don’t leave the light on too much or you’ll kill your cells! Definitely avoid using UV sterilisation lamps; stick with the sort you use to check banknotes, which use lower frequency light which is less harmful.
If you get your desired glowing cells, congratulations! Reunite them with L.bulgaricus, and try brewing up your first batch of fluorescent yoghurt! If not, try again and don’t worry too much. The transformation frequencies with S.thermophilus are really low, so perseverance may be called for.
However, in the end of the day, it mightn’t work at all, and there might be no foreseeable reason why it didn’t. If so, you’ll have joined the ranks of biohackers worldwide who’ve tried only to meet with unexplained failure. I hope the project didn’t cost you too much to attempt, but don’t throw it all out; share that DNA with others, see if they can improve the procedure; perhaps a different insertion site? A slight modification to the integrated DNA? A different transformation procedure? Most importantly of all, share your failure; mankind learns most from failure, and not enough failures are shared in science. Be the difference, speak up and tell your cohort what failed for you!
If you want to try this, get in touch with me first for more in-depth advice on some of the details. Before you commit, bear in mind that this project will cost a lot of money at current costs per nucleotide! Unless you can access the DNA for a green fluorescent protein and a resistance cassette (which would save you a lot of synthesis money), you’ll have to order these from scratch, and that won’t be cheap. At least a few hundred Euro. However, the reward is something you can share with others and boast about for years to come; your own fluorescent GMO yoghurt. Very 2012.
By now, you’ve probably heard of the “Stop Online Piracy Act”, a doublespeakish attempt to permit massive censorship of the Internet by private corporations and shady government agencies without oversight or dispute in the USA. Under SOPA, it would become a felony to even link to a site that has been censored, and censorship would occur at the financial level (akin to the illegal financial blockade on Wikileaks.org) and the DNS level (i.e. domain names would cease to work).
I subscribe to the view that, while combating such attempts through political and legal channels is essential, there must be another current of resistance in the form of practical effort. And the best two efforts I’ve seen so far are Tor (The Onion Router), a secure and anonymising routing system that protects the identity and location of web users while enabling them to access otherwise censored content, and a grassroots effort by reddit users to assemble a list of IP addresses for websites at risk of censorship in the USA. Indeed, I’ve added a slightly altered version of this list in my “Links” section, which I will probably expand with some static IPs matching my favourite DIYbio sites in due course.
Tor is a tremendously sophisticated way for people to access censored content and to preserve their identities as they do so, but Tor is itself susceptible to censorship by forbidding users access to the Tor network in the first place. In order to avoid this, Tor users provide “bridge relays”, entry nodes which can allow access to the network without appearing on an easily censored list of relays. However, censors have become more sophisticated at detecting and rapidly banning bridge relays, so more effort is needed.
A promising new approach appeared recently in the form of browser-powered, transient “Flash-Proxies”, which are fired up as users browse webpages with Flash Proxies embedded within them and redirect censored users to Bridge Relays in a way that is hard to detect and censor.
The beauty of this approach is that browsing web users become transient proxies for access to Tor relays as they browse, clicking in-and-out of the proxy network as they browse around. It would be extremely challenging to censor these Flash Proxies without censoring broad IP address ranges and crippling a country’s network access to anything.
The system isn’t perfect and it still has a few weak points, but it is already ready for testing and contribution. To this end, I have embedded a Flash-Proxy into the sidebar of Indiebiotech.com; while you have been reading this, in all likelihood your browser has been acting as a Flashproxy. Did you notice? Probably not. It’s that little “I support Internet Freedom” icon on the right. If you object, you can disable flash while visiting, or disable “iframe” elements using something like the NOSCRIPT plugin for Firefox (or even specifically ban the FlashProxy plugin using the same).
So there you have it. The reason I’ve been discussing censorship on a biotech site is to inform you that you, my visitors (however few you may be), are now complicit in preventing Internet censorship for so long as you remain on my site. Thank you for that, and I hope you don’t mind having done so.
Here’s a belated notice for those able to make last-minute flightplans. This weekend, the Manchester Madlab are hosting a DIYbio Summit, and the lineup looks great.
You can book places for the DIYbio Summit on Eventbrite; please come if you’re able, there’s already a great crowd due to attend. As for Indie Biotech, yours truly will be giving a 30 minute keynote on the first day. I’ll also be helping to facilitate several workshops over the weekend, with plans ranging from in-silico gene design (or, “How do I go from Concept to Ordered DNA?”), a rundown on Irish GMO Licensing Law (which is rather similar to the UK system), and assisting Brian Degger of Transitlab.org in giving a Webcam-Microscope hacking workshop.
At the unconference on day 2, I also have loose plans to share some protocols I have used successfully in the past year that make biohacking and DIYbio a lot easier/safer, and to share and air some ideas for feedback or collaboration.
Looking forward to seeing you there!
A twitter acquaintance asked me today about DIY thyroxine. As this acquaintance is a “collapsonomics” nerd, I took this question to be in the context of “what if the world’s supply chains freeze and I/we/my friends die?”. The question broadened to include insulin, another critical drug needed daily by those with a common condition, diabetes.
That’s a fair question. After all, while one can grow food locally, and purify rainwater (when blessed by rainfall like we are in most of Europe), and tend to many other necessities of life locally in a total infrastructural breakdown scenario, pharmaceuticals are one of the few things that we can’t DIY easily. As a fan of the simplicity and focus of Vinay Gupta’s “Simple Critical Infrastructure Maps”, I imagine the problem as being an outlier in the “illness” sector of a person’s SCIM. Specifically, the provision of essential drugs in case of collapse probably normally approaches the person (in the center of the map) via the local hospital or pharmacy at the town/city circle, but the actual source of these drugs is likely to be in the “national” or “international” circle; very far away and very inaccessible in case of emergencies.
And these emergencies are not imaginary. Just this year, an entirely unavoidable natural disaster claimed Northern Japan, rendering a confident, successful capitalist state into a disaster zone for days/weeks. People in the disaster area were without critical infrastructure, and no doubt some still are. Haiti suffered complete collapse after another such natural disaster; you might think “they were poor already” but Haiti was nonetheless a country with infrastructure before the collapse. Now, they’re suffering a cholera outbreak and a truly huge proportion of their population that survived is homeless and without infrastructure still.
Just because it didn’t happen to Europe or America yet, doesn’t mean it can’t. Much of both super-states lies on or near fault lines, tidal zones, or rapidly changing climate zones. And that’s *just* dealing with natural disasters, when in fact economic disaster has been looming for years now on both sides of the Atlantic. Provision of insulin and thyroxine depends on functioning infrastructure and a market system that rewards large biotech companies for making and selling them. Infrastructure and Markets can be destroyed, often without warning, by extrinsic or intrinsic factors.
So what can you do to prepare for the worst if your continued living depends utterly on Insulin or Thyroxine? You can either stockpile the drugs, or you can work out ways of producing them on demand if and when they’re needed. The latter has the benefit of being scalable to meet unanticipated demand; if you’re the only guy in town with insulin and the whole town goes to hell in a basket, you can share a production platform, but sharing your stockpile won’t go very far.
My method of choice for sustainable and resilient design is biotech. That’s no secret at all; I founded Indie Biotech because I hope that one day we’ll all have the power to produce life-changing things with the most natural engineering platform ever devised; the living cell. Insulin is already produced by transgenic cells; the problem is that these cells aren’t in your possession yet, and so you have no power to produce your own. Both insulin and thyroxine can be made DIY with the right genetic program. However, there’s a caveat.
And that caveat is that, unless you have some astounding equipment on hand, you almost certainly can not prepare injectable drugs of any kind safely. That’s because your production platform is probably going to be a microbe, and the body does not take kindly to microbe-parts being injected into it. Indeed, if you use E.coli to produce your drugs, you’ve got things like Lipopolysaccharide (LPS) to consider as contaminants; your odds of DIYing an LPS-removing production method are next to nil, and contaminating LPS can cause a lethal immune response.
Therefore, if you’re planning to make thyroxine or insulin, you’re planning to firstly produce it through as safe a process as you can manage, and secondly consume it in a manner that doesn’t pose an immediate, lethal immunogenic hazard.
I should point out at this juncture that it’s a severely bad idea to try and make drugs yourself and self-administer, particularly with drugs that can be lethal in the wrong dosages like Insulin and Thyroxine. If you can get critical medicines through any conventional channel, you should use those channels. Trying to make your own just for the thrill of self-sufficiency will probably end very sadly for you. However, if you were a diabetic stuck in a disaster or collapse zone with no source of insulin, it’d be nice to have a DIY production platform before you run out of normal stocks and die.
In brief, I’m trying to say: this is a theoretical discussion, right here. I’m not suggesting or endorsing that anybody should make medicines at home and dose themselves, and I encourage people to check the facts for themselves and consult a professional before they attempt anything of the sort. If I were to say otherwise, I’d be liable for hefty lawsuits and might even spend time in jail. And now you know why there aren’t any Edward Jenners this generation.
So without further ado..
Insulin is a peptide hormone, produced by the islets of the pancreas from a normally expressed protein by extensive post-translational modification. Specifically, it’s produced first by the ribosomes attached to the Endoplasmic reticulum into which it is synthesised. Within the ER, its cysteine residues are cross-linked (passively, by the redox environment) into a set of disulphide bonds. Once folded and bonded in this manner, bits of it are cleaved by a set of enzymes until the mature hormone is created and secreted. The hormone is hexameric; that is, six subunits of mature insulin bond together with help from a zinc ion to form the final, functional hormone.
The critical genetic parts needed for producing insulin are the proinsulin polypeptide, prohormone convertase (AKA neuroendocrine convertase) 1 and 2, carboxypeptidase E, and a bunch of specific genetic functions for your production platform that facilitate proper folding and secretion.
Most of the latter; that is, the programming end of production, is generally pretty small compared to the size of the coding region of a gene. So, to calculate roughly such things I normally estimate +15% on top of the protein-coding portion of an operon. However, with Proinsulin being comparatively small as a coding region yet requiring just as much regulatory fun, let’s assume 20% regulatory DNA, and estimate price.
A jump to Uniprot and a search for Human Proinsulin, PC1, PC2 and Carboxypeptidase E reveals the following sizes in terms of amino acids:
Human Proinsulin: 110aa
Human PC1: 753aa
Human PC2: 638aa
Human Carboxypeptidase E: 476aa
Totalling those gives 1977 amino acids. For each amino acid, per the amino acid code, three nucleotides are needed; that means three units of DNA to each unit of polypeptide. That gives 5931bp. As I decided to go with a 20% regulatory burden, we’ll add 20% to that, giving us 7117bp (dropping decimals).
At roughly current costs of gene synthesis (which are falling at a decent pace, mind) of €0.29, a total synthesis of the necessary genes to make insulin would cost about €2,064.
The reason I went with 20% and didn’t bother estimating any more accurately is because the regulatory DNA is the highly variable bit, depending on your expression platform. That is, do you want to make this in E.coli (easy to make, strong toxic response), Yeast (harder to design DNA but more biocompatible and more advanced secretory machinery), or Bacillus subtilis (somewhere in between on both ease of design and likely biocompatibility), or something else entirely? The answer to that question will decide how your regulatory budget of 1186bp is spent. You may end up using more or less; each BP added or removed from the budget carries €0.29 with it.
Once you get your DNA designed and synthesised, and it arrives in the post, you’re aiming to get it into the target species and to produce insulin at a useful level. How you’re planning to do that feeds back into the above question about how to spend your regulatory budget.
Firstly, how are you keeping the DNA in the cells? If your DNA is free-floating in the cells as a self-sustaining system (for example as a plasmid), you need to ensure that cells don’t abandon the DNA because it offers them no survival advantage. If the DNA is somehow integrated into the chromosome of the cells, this is less of a problem. Forcing cells to keep the plasmid is usually achieved with antibiotics; that’s something I’m aiming to undermine with more friendly methods in Bacillus subtilis, but it’s nonetheless the industry standard. That presents a problem to off-grid production of drugs, because it requires other drugs. Here, clever engineering is called for that is outside the scope of this already messy essay. Suffice to say there are many options.
Secondly, how are you planning to prepare and use the product? If your cells are producing the insulin inside themselves and keeping it there, you’ll need to extract the insulin somehow. Again, outside the scope of this discussion. If the cells are exporting (secreting) the insulin, either before or after maturation by the PC1/PC2/Carboxypeptidase set of proteins, then you can extract the insulin from the media surrounding the cells..but you’ll have to ensure that other exported proteins don’t either destroy the insulin or contaminate the product some other way. For example, without more DNA reprogramming to prevent it, Bacillus subtilis produces a cocktail of extracellular proteases; enzymes that eat other proteins and polypeptides, like insulin.
Once you’ve sorted out your DNA code, your expression platform, and your system of production and purification, you need to consider how it’s getting dosed. For insulin, practical routes include anything but oral delivery; your stomach proteases destroy insulin quickly on ingestion. Nasal and inhalable routes are possible, though the preparation is important toward bioavailability. You need to consider and research whether these routes will tolerate much contamination; your lungs mightn’t like Bacillus cellular matter much more than your veins, for example. The level of purity required will depend, therefore, on the mode of delivery.
So that’s a rough outline of what you’d need for insulin, and what engineering challenges are faced. Thankfully, the following is less medically complicated, though no less technically challenging.
Thyroxine is normally produced by a pretty complex route in the thyroid gland. It’s not a peptide hormone like insulin per se, but rather a fragment of a modified peptide chopped off of a larger protein.
Much of the complexity in the thyroxine synthesis pathway/system comes down to transportation of thyroglobulin (which might be considered “pro-thyroxine”: it’s the protein from which matured thyroxine is cleaved) and iodine in and out of cells in the thyroid. These complexities aren’t quite so relevant to a mono-cellular production system, so they can be ignored.
In brief, the production of thyroxine follows this course: ionic iodine is brought together with thyroglobulin, and is then oxidised to nonionic iodine by thyroperoxidase in association with hydrogen peroxide. The highly reactive iodine reacts with tyrosine residues on the thyroglobulin protein, cross-linking tyrosines with iodine atoms. The thoroughly cross-linked set of tyrosines on thyroglobulin are released by proteolytic cleavage (in other words, after being eaten by protease enzymes) to yield the “T4″ form of thyroxine, which is released into the blood. T4 is converted in target tissues to T3, a more active form of thyroxine; T4 is considered a prohormone.
So, the critical machinery for production of Thyroxine is (sizes from Uniprot.org, again):
Thyroglobulin (2,768aa),
Thyroperoxidase (933aa),
Assorted Proteases (Unknown; potentially part of production chassis already?)
Cofactors include:
Iodide (I-),
Hydrogen Peroxide
Looking at this and knowing what I know about B.subtilis, I’d suggest using that bacterium for thyroxine synthesis. B.subtilis can theoretically export both critical proteins outside the cell environment, which is probably critical to their oxidative function, and in later stages of growth B.subtilis expresses a host of potent extracellular proteases that could finish digesting thyroglobulin into mature T4 thyroxine. As Thyroxine can be taken orally and B.subtilis is not only edible but very possibly good for you, overall this combination of factors leads me to favour it for thyroxine biosynthesis. It’s also really easy to grow at home and maintain stable cultures of at room temperature.
The total amino acid count in this case is 3701aa. Multiplied by three, that gives 11,103bp of DNA. Adding 15% regulatory code gives 12,768bp. At a cost of €0.29/bp, that gives a rough price estimate of €3702.85.
The feasibility of production is probably greater here than with insulin; once produced by crosslinking and cleavage of iodised tyrosine, the resulting hormone is much tougher and can be eaten. Indeed, thyroxine used to be produced by grinding and pill-forming animal thyroids, which would make a feasible fallback if you can’t biosynthesise human thyroxine in an emergency.
Therefore, I imagine synthesis looks like this;
On-demand biosynthesis of medicine in deprived regions is something of a pipe-dream of mine. I want to see it happen, because most of the world already live in a state of constant crisis and those that don’t are at risk of it. Insulin isn’t just an occasional life-saving drug like antibiotics; it’s utterly essential to continued life for those with diabetes. Likewise for thyroxine for those with severe hypothyroidism. Producing these medicines for those who need them is a critical task for society; if that breaks down, what are the alternatives? At present, there are none. This is one potential avenue of survival, but it requires preparation on the part of those who want to see it happen.
It doesn’t end with thyroxine and insulin at all. In fact, it only began with them, two and a half decades ago with the biosynthesis of insulin in genetically modified bacteria by Genentech. Biosynthesis of drugs and drug precursors is now routine and is becoming more critical to the way we provide medicine to the first world, but it’s controlled by vastly powerful but incredibly distant organisations that won’t be there if everything goes to hell in a handbasket. They’re already not-there for billions of people worldwide.
If more critical drugs are targeted for on-site production by bacteria or yeast, and if small-scale production and dosing platforms can be devised for them, the impact will be immense. Biosynthesis doesn’t need any infrastructure that isn’t readily available almost anywhere (albiet to those with money to acquire them); pressure cookers to sterilise things, containers, basic food ingredients to feed the bugs. Centrifuges made from egg beaters or dremels optional. Once made, a bio-production platform is just another culture to be shared between persons, villages, and organisations for the betterment of the planet.
That’s what Indie Biotech is ultimately about. Making Biotech a household appliance for lifesaving and world-altering applications.
I’m a serial blog-abandoner, but I’m generally good for eventually returning to them and reviving them again for a while. Consider this an update since the last post, in which time much has happened and more is expected soon.
Workshops
The science gallery workshops went great, as far as I’m concerned. The (regrettably few) participants were great fun to work with, and I learned a lot about the unique requirements of a mobile or temporary lab. I’m getting there, slowly formulating a “protocol” for a rapid assembly minimum lab for synthetic biology work.
It’s a pity that the timing of the workshops was so bad, though; more lessons learned there. Conflicts with visiting pop-idols meant that transport and accommodation was too expensive for some excellent people to make it, and there wasn’t enough time to really spread word of what was happening; on the imaginary “hype curve” the workshops happened maybe halfway up the climb, too early for many enthusiasts to hear about it or make preparations to take part.
I’m hoping that for the next workshops I run I can offer more time, and be more flexible on the timing so I can entice as many inspiring souls to join me as possible. More on that soon.
Protocols and Informational Stuff
There’s a whole section on the site here with supposed protocols, but no links to those protocols. Have had a few comments about that, and it’s a minor private shame until it’s done. The reason why those articles are listed but not linked was because I was planning to blitz through writing them in short order, but got interrupted repeatedly.
Also, I thought I could log in at home and write them in spare moments, but discovered an oddity; the craptop I use for work in my home can’t handle WordPress for some reason. Everything renders, but writing is impractical. The fact that it can handle Google Docs but not the text input of WordPress puzzles me. Add to this a recent complete-device-failure of my desktop, and I’m hoping you can learn to forgive me for being late with those articles.
They’ll start appearing soon. I’m writing “How to set up your home lab”, and I have documents I can adapt easily enough for “Isolating bacteria X” or “Growing B.subtilis” or “Extracting plasmids from Yoghurt bacteria”.
EPA License – Check
Big news for me is that I’m now officially licensed for “Contained Use of Class 1 GMMs” by the EPA. I’m hoping to share a post soon on my experience in getting a license, subject to some omissions and tact, to show from a first-hand perspective how much of an uphill struggle it’s going to be to help encourage citizen science in Europe. Ireland is one of the less hostile countries to modern biotechnology, and it’s still difficult to get a license and to adhere to the conditions of it once received.
Suffice to say I’m still doing the paperwork and fulfilling the special requirements of my consent conditions, so work has not yet started. Somewhere between now and soon I’ll start writing the process; both as a how-to and as a mildly disheartening look at the state of biotech regulation in Ireland.
It’s not too late for you to take part in the biohacking workshops that I’ve been facilitating in the Science Gallery this past week. It’s true, the wet-work is officially out of the way, and tomorrow’s session assumes some grounding that has been provided in prior days.
However, the last day will focus not on the methods, nor even the knowledge of biotech. Instead, we’ll focus on the role of biohacking in society, with a particular focus on the transformative power of DIY biotech, the social responsibility and ethics that this calls for, and the particulars of Irish law regarding biohacking.
I would also like to see a discussion on the social aspect of DIYbio in Ireland; to gauge interest and capacity for social DIYbio meetups, events and group projects, and perhaps even someday to found a “bio-hackerspace”, where the still-noteworthy costs of establishing an indie lab are shared, and expertise and enthusiasm are pooled.
If you are interested in joining us this Saturday from 1PM to 4PM, give Maria at Science Gallery a call on 01 896 4107 and ask about the one-day rate for Saturday. I’ll be sincerely glad you came!
Finally, let me take this moment to offer my thanks to the staff at Science Gallery; they are wonderfully helpful, enthusiastic and engaging people, and I’m really enjoying working with them.