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.

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