Letters

Hello Makerbotters and curious folk. Here's a well deserved update for the "Mouse Get!" Challenge I posted.. er.. ages ago.
To recap for those unaware: the challenge was to create a printable, live-catching mousetrap that could be made with a Makerbot. That worked. I offered $25 for the winning design. Makerbot Industries added an optional condition that the winning trap be Open-Source, which was an obvious condition that I somehow forgot (d'oh), and awesomely added a Teeshirt to the prize.

Of course as I now realise, it was foolish to undertake such a challenge without more conditions at this stage in personal-scale 3D printing. I should have asked that prints be no larger/longer than X, or that only the first three entries would be considered. Or that I could curate the submissions and pick three!

I say this because, printing is not yet foolproof and easy. No sooner had I posted the challenge in fact, than I suffered the worst breakdown I've yet had and needed a replacement plastruder (which works really great, thankfully.. but is now showing signs of wear also! O_o). Long prints were prone to failure if the bot did anything untoward, wasting perhaps an hour's printing. Plastic requirements were high, and I needed to order some more after the batch I had gotten from a local supplier turned out to be a dud. SD cards died and wasted time, computers died and wasted time, and software/firmware issues bugged me along the way.

All of this, and I also had a wedding (mine!) to experience, a Honeymoon to enjoy, and a Masters project/thesis to do (ongoing! ).
When I have had time to Makerbot, I have endeavoured to print at least one Mousetrap each time, although I have had other makerbotting responsibilities such as a long-promised repair job at work and some spare insulator retainers for my friends at the Tog Hackerspace. After printing out a full printruder that is, just in case.

Those feeling let down by the pace can rest assured that I am frustrated also. But, it's not all bad. I have printed the Claptrap, and await further instruction from WorldGnat before I test it (because I don't know how he intended it to be used). I have printed the Tower Bridge trap from Starno, and it's in front of me now. I'll share a picture here. It's awesome. I printed and tested the Merry-Go-Round trap, which sadly didn't work (though I reckon with a redesign, it would!). And as I write this, I am preparing to try printing the next trap in full: The Mouse Motel from TeamTeamUSA.

Sorry again, as I've been saying for too long. I can't wait to be done printing these traps and testing them. The designs are really amazing, even when they don't work. They show imagination and resourcefulness that inspires me in my own designs, and I consider each a victory even when no captive mouse results.

Posted via email from Cathal Garvey

DIYBIO4Beginners is over a year old, and has a huge number of posts covering mostly practical aspects of learning about Biotech and DIYbio. Of particular interest are the posts that contain video series' teaching you about Biotech/Biochem, or "How-To", etc. There's a lot of stuff in there, much of it news coverage, but the signal-to-noise is really excellent. I'm gonna have fun reading the archives.
Some highlights: Eric Allen's Biochemistry Lectures, Prof. S. Dasgupta's Lectures on Metabolism, DNA/Protein/Enzyme Structure and Kinetics.

Another great blog focused entirely on the practical side of things is Citizen of Science, and I particularly like how the author puts a lot of thought into recycling and upcycling household materials into labware. An example is his use of old lightbulbs as boiling flasks. He also made a centrifuge out of a blender, and a cell density meter using a solar cell and a bright light source (I'll probably have to do something similar with a photocell soon; Spectrophotometers are simply too expensive!).

I also sidetracked myself learning about how to get stuff (TM). Among my discoveries:

• You can get almost anything from alibaba.com, although up front pricing is not often available. I've found tens of suppliers for high-fidelity polymerase, agarose, Ni-sepharose, etc. I asked about pricing for Ni-Sepharose, and I hope to be pleasantly surprised. For the uninitiated, Ni-sepharose is a special resin used to purify transgenic proteins quickly and easily, but it's usually super-expensive.
• http://www.mistralni.co.uk/ and http://mistral.ie/ for lab-grade chemicals, solvents, acids and alcohols. In keeping with the silliness of law, you can buy exceptionally toxic chemicals from them but not Ethanol. Of course, the exceptionally toxic ones aren't habit-forming and are unlikely to lead to domestic abuse, but labs need pure ethanol for good reasons sometimes y'know. Still, these sites are selling affordable chemicals inclusive of VAT and shipping to Ireland, and accept Paypal. I love them already.
• More on the chemicals front; in case you've forgotten or never encountered it, here's a great resource on Makezine.com detailing the essentials of a good chemical lab, including ways to quickly synthesise loads of essentials not readily available. Much of this is only applicable to the USA, because over here things are a little harder to find pure sometimes and because our brand-names for household chemicals are different. Non-denatured Ethanol being an example. But it's still awesome. And it's part of a series that may contain other gems, even if it's not targeted at biological labs.

I have other cool things to write about soon. For now, this will satiate me.

Posted via email from Cathal Garvey

DIYbio and its more professionally oriented cousin, Garage Biotech, are undergoing a revolution at present. Essential equipment that used to cost thousands is now available at affordable prices, in many cases under open licensing schemes and open to community development. Knowledge of biology, genetics and the procedures underlying it all is being disseminated in ever-more-abstracted forms to make it easier to get started. And soon, even the biological components: strains, enzymes and substrates, will likely become mass-marketable.

It’s an exciting time to be involved in the development of tomorrow’s technology, and sometimes I find myself stepping back to consider what we have, and what we still need. I may as well share these musings with others to spare them the time, and perhaps to inspire someone with the know-how to fill in the gaps and help make this happen.

Prepare for a long, long post.

Equipment

Basic Liquid Handling – Have!

Although there has been no obvious effort to generate an Open-Hardware version of these tools, micropipettes are now available inexpensively on Ebay and Aliexpress. Likewise, bulb-type pipette fillers (rubber bulbs that you squeeze to fill and manipulate rubber valves to empty) and plunger-type fillers (roller syringe-arrangements) are available from ebay and aliexpress.

There is often talk of making a liquid handler robot; the most likely candidate would be a modded Makerbot, perhaps a drop-in liquid handler that sits on the Z-stage instead of the makerbot plastruder module. Nobody has yet designed a feasible, affordable liquid handling system for DIYbio, but it probably won’t be long. The next question is how necessary such a system would even be for most individuals, but we’ll leave that unasked for now.

Sterilisation – Have!

Autoclaves are too big and too expensive for individuals to buy or build. Community design of an autoclave is unlikely to happen, because of liability and safety issues. Thankfully, autoclaves are just giant pressure cookers; use of a normal culinary pressure cooker is sufficient to sterilise things well enough for science. One might require several pressure cookers to meet demand in an active lab, however.

The problem arises with larger containers and tools, such as pipettes and 500mL-1L erlenmyer flasks, etc.: these simply will not fit in a culinary pressure cooker! Although larger versions can be had to fit such equipment, they are sold as “Lab equipment”, which naturally means the suppliers start to charge ludicrous margins at that point.

Use of concentrated bleach is supposedly an effective means of sterilising glassware such as pipettes, although it poses a unique safety hazard and disposal issue. Also, the unwitting may think to mix their bleach, peroxide or solvent waste which is a bad, bad idea. Keep chemicals separate, people. However, when needs demand, a bucket of bleach will do for sterilising awkward glassware, and a quick rinse with sterile water and denatured ethanol will prepare them for use: just keep them in a container you’ve sterilised also (perhaps an autoclavable plastic bag that you can seal?

Aseptic Equipment – Have!

The natural continuation of sterilising things is keeping them sterile; in labs, either bunsen burners or laminar flow hoods are used to keep work sterile. The former works by establishing a closed convection loop of sterile air, and bunsen burners can be found that are simply mounted onto camping-burner cardridges.

The latter can be simulated by placing room-scale HEPA filters such that the outflow blows directly downward onto the work-bench, but issues with things blowing away may arise. A creative person might create a DIY laminar flow hood from modified furniture, somewhat like mushroom-growing hobbyists do already, or simplify their job even more by using a room-air purifier and a cabinet. Make sure to get a good brand that has a very fine filter, because ideally what comes out the other end is absolutely free of spores.

Whether using a bunsen or a hood, it makes sense to have an air purifier or two working in your labspace anyway, to keep the ambient air fairly clean to begin with. The less particulate matter might screw up an experiment in the absence of aseptic equipment, the less likely your hacked aseptic hardware is to fail you.

It also makes sense to have UV lamps fitted in your labspace and inside your hacked flowhood. When you are leaving the lab, put these UV lamps on a timer for an hour or two to sterilise surfaces and destroy any stray bits of DNA that might be hanging around. These guys can come back to haunt you by contaminating cell transformations later on, and UV lamps are a cheap, sustainable way of keeping your surfaces clean.

Finally, if you’re dedicating a room to your biotech hobby, it would make sense to dress appropriately. Wear low-dust clothes, wear a natural-fibre* labcoat over this, and tie back hair or wear a hair-net. Obviously, always wear clean gloves; sterilise with a 70% ethanol spray or an alcohol-based antiseptic hand gel.

*Natural fibres are a better idea where there is a risk of ignition, like in a lab, because they do not shrink and stick to you when they catch fire. Use a cotton labcoat with press-snap buttons so it can be torn off in case of chemical spills or fire.

Incubators – Don’t Have!

These are large, expensive and cumbersome. They are also unlikely to be easily produced to a fully satisfactory standard by the average hobbyist. Fortunately many bacteria do not strictly speaking need incubation, and it is likely that whatever organisms become the norm for DIYbio will be among those that lack a strict temperature requirement.

If you do want to grow bugs that prefer the warmth, consider Aquarium supplies your friends. Many aquarium heaters will heat water to a little above 30C, and even this “limit” can be worked around if one bought a tunable heater. Be sure to couple the heater with a small water pump to homogenise the temperature, and you’ll have a reasonably reliable water bath at incubation temperatures for growing cultures. This is obviously better for flask/tube grown cultures than petri dishes.

One way I can forsee for the community to develop an easy-to-use and easily-installable incubator is to create a circuit board that uses a trace heater element like that on the Makerbot heated build platform, with two small dials; one for temperature, and one that chooses roughly the volume of the area to be heated to help define PID parameters in the arduino firmware. The board would then be attached to an arduino, powered by a micro-ATX supply, and simply placed in the volume of space to be heated. The trace heater on the makerbot board can exceed 100C, so this should be more than sufficient to heat a chamber to incubation temperatures! I ought to kickstart this discussion on the DIYbio group.

Thermal Cycler – Have!

Until recently, it seemed every third person involved in DIYbio was personally dabbling in thermal cycling, trying to recreate their own peltier-thermal cycler at home. I made my own stab at it a few months ago.

Thankfully, some geniuses (Tito Jankowski and Josh Perfetto) in San Francisco have founded OpenPCR and are actively prototyping an Open-Hardware design for a peltier thermal cycler, which they will then be selling for (planned) $400. The design currently accepts 16 tubes, and they claim a ramp speed of 2C/s, more than many commercial machines and more than sufficient for PCR (in fact, many protocols call for deliberately slower ramp speeds..).

I’ve already ordered one through the OpenPCR Kickstarter fundraiser, and hopefully in a few months I’ll have a working DIYbio-built thermal cycler to test out.

By way of making great connections where they are due, these guys are also heavily involved in setting up and fundraising for the very amazing Biocurious, perhaps the first dedicated hackerspace for Biotech. They are also fundraising on Kickstarter for this lab, so if you like the idea go help out!

Gel Electrophoresis – Have!

Thanks again to the good folk of San Fran, because Tito Jankowski and Norman Wang are selling an excellent electrophoresis kit through Pearl Biotech. This is a great example of a superior product from the community, because I’ve never seen a comparable offering from industry.

The Pearl gelbox requires a separate power supply, and a camera to take pictures of the results. But aside from that, it’s an all-inclusive design that marries band visualisation to actual migration of DNA, and allows you to cast gels in the rig simply by rotating the gel-boat by 90 degrees (the rubber gaskets at the ends of the boat form a seal). Because it’s designed specifically for Sybr-Safe, it comes with a blue transilluminator and an orange filter. In theory, you could simply leave the blue lights on and watch your bands migrate in real time!

And, it’s only €200 or so for a mostly assembled kit, requiring only basic soldering.

Centrifuge – Have!

Although second-hand centrifuges can be bought on ebay, they are often still very expensive and very heavy to ship. For a dedicated hobbyist, it’s probably a worthy investment in terms of capacity and safety.

However, for those on a budget who are willing to take the risk, I designed a centrifuge rotor that is mounted on a Dremel tool, which I called (aptly I think) “Dremelfuge”.

Dremelfuge is pretty hazardous, in that it’s a naked rotor spinning up to six tubes at up to 33,000RPM (which is 52,000g, by the way). But, for those willing to take that risk, it’s a functional centrifuge for sale on Shapeways for under €70 (costs may vary by location, so I don’t know what price you’ll see. I see €54).

There is also an edition with a post for a chuck instead of a recess for a dremel; just in case you would rather spin it with some other tool. I haven’t tested the chuck version on anything significant, so I can’t speak for its reliability or safety.

Protein Purification Apparatus – Don’t Have!

Extracting and purifying proteins from cells is sort of necessary for effective production of things like enzymes, which might be a garage requirement for people who can’t afford expensive enzymes, or a critical part of research.

The options are many, and are summarised pretty well on the wikipedia article on protein extraction. However, unique requirements of each protein mean that a single, easy method for all use cases is unlikely. Perhaps the easiest answer for most of us would be a homebrew solvent-based lysis buffer followed by centrifugation to remove cellular debris, but the supernatant will contain all sorts of proteins including proteases, RNAses and perhaps DNAses which will post obvious problems for extraction of DNA-altering enzymes.

If the protein is transgenic, then things are a little easier; make sure you attached a His-tag, and buy some centrifuge-friendly nickel-affinity columns. Figure out how to equilibriate, wash, elute and clean the columns and they’d probably be OK for re-use.

What is needed here, however, is a robust, DIY-friendly chromatography rig or an alternative protocol that works for a majority of cases. Perhaps a system based on electrophoresis of protein would work nicely for those who already own a gel rig (see above). Perhaps a differential centrifugation step for those with a centrifuge. It’s unlikely to be a clean-cut part of the hobby..

Storage – Don’t Quite Have..

So, we have -20C freezers in most houses. It’s easy to freeze stuff, but make sure to buy a frost-free freezer and keep cold-packs in there to buffer your important samples against freeze/thaw damage. However, things aren’t as simple as just freezing; there’s different kinds of freezing, and cells require the most annoyingly precise freezing of all.

Although proteins are stable at -20, cells like to be kept at -80. The reason for this is that lethal ice crystals are less likely to grow and lyse cells at lower temperatures, where water ice takes on a different structure. In order to better protect cells, we usually freeze cells with “cryoprotectants”, the most common and easily accessible of which is glycerol at upwards of 40%.

There is an experiment ongoing by one helpful member of the DIYbio group into small ceramic or glass toroid beads, of the sort that you thread necklaces with, in combination with glycerol, as a storage medium for -20C cell stocking. Cells are scraped from a plate, suspended in a little liquid, and shaken all over the beads. Then excess liquid is withdrawn leaving a cell-rich film over the beads.

This is based on my observation of a fellow microbiologist storing her stocks in a commercial kit that is based on small ceramic beads of this sort, and our hypothesis that the curved surface of the beads helps to prevent ice crystal formation. This method, if it works, would be an excellent option for DIYbio, because the ingredients and requirements are very easy to achieve.

Finally, if you really need -80C you can probably fake it by keeping a well-insulated polystyrene box in the freezer and topping it off with dry ice beads every few days. You’ll need a supplier of dry ice and the commitment to constantly make sure it’s not evaporated away. You’ll need a lid to prevent the comparatively toasty -20C air from evaporating all your dry ice. Not a screw-on tight lid, because then your container might explode, but a lid that will keep the air inside static.

The other option is pricier but likely to work well once established; use of a hard vacuum to lyophilise cells into a powder. I am led to understand that this is a highly reliable way of storing cells, but it requires a powerful vacuum pump and ideally a -60C/-80C stage between the cells and the pump where water vapour can condense before it corrodes the pump’s innards. You could achieve this with a bell jar and some dry ice, I suppose, but the cost of the vacuum and the hazards of a hard vacuum give me pause. For long-term storage, there’s no contest, but it might have to be done by DIYers by asking a favour of a local microbiologist with a professional rig in their lab.

Spectrophotometry – Don’t Have!

Measuring DNA, RNA, Protein and Cell Optical Density requires a spectrophotometer or spectroscope. The absorbances vary between 230nm and 320nm, and the equipment is still very expensive second hand.

In principal, a community-built “spec” shouldn’t be too difficult to design; you need a UV source (a bulb?), a light path through a UV-permissive cuvette in which the sample is diluted, and a range of photodiodes that respond to specific frequencies. Or something like that, I’m only passably fluent in electronics. Point is, it shouldn’t be hard to design, so I think it should be an area of active focus for those involved in DIYbio who know more about electronics.

Without a working spectrometer, measurements of DNA quantity can be roughly made by comparing a sample on a gel to a standard of known concentration/intensity. I can’t offhand think of a convenient way to check the OD of a bacterial broth, though I ought to disclose that in my time working in a lab I’ve never had to. But then, I’ve never grown anything for reasons other than plasmid preparation, and proper rigorous study of a transgenic microbe vs. the wild type would call for growth-curve analysis by OD measurements at defined timepoints. Without a spec, you simply can’t do this sort of science.

Consumables

Broths for Bacteria – Have!

Although you can buy broth powders on ebay, you can also make bacterial broth/media yourself using off the shelf ingredients. It’s satisfying and fun to do, and costs barely anything! I used it to successfully isolate and culture the glowing bacterium Photobacterium phosphoreum, and it worked out very well.

So if you’re too cheap to buy the readily available powder, you can always make your own.

Chemicals and Solvents – Varies

Depending on your country, you can either easily source solvents and chemicals for essential tasks in the lab, or you can’t. Oddly enough, one of the least accessible solvents is also one of the most necessary and least toxic; Ethanol, or “drinking” alcohol. This is because (surprise surprise) there are many idiots who would drink themselves to death immediately if they could get high-purity ethanol. Especially where I’m from.

The easiest way to find out whether it’s legal to purchase certain chemicals in your country is to call a supplier and ask them what is required from their buyers to satisfy legal requirements. In Ireland, it turns out that you need only be a private company to qualify for purchasing any chemical required for biology. I consider it highly ironic that it is easier and cheaper to purchase potentially lethal high-molarity hydrochloric acid than to buy ethanol.

Disposal can, annoyingly, be more of an issue than procurement. Post-biotech chemicals pose a serious ecological hazard and may not be legal to dump or drain where you live (neither should they be). You will probably have to collect your chemical wastes in inert plastic containers and contract your supplier to dispose of them safely. Remember to pay close attention to what waste goes where; mixing things like bleach and acetone can be a recipe for disaster. Best to keep your solvents separate where possible, which may even aid the disposal crews in recovering and recycling solvents in some cases.

There is a need in DIYbio to establish exactly which solvents are surplus to requirement, however; many solvents are considered necessary for routine tasks in the lab, but is this because they are indispensible, or because they are the most convenient option in labs with access to them? What can we do without or substitute? Some time ago, Meredith Patterson brought up the possibility of doing a Phenol/Chloroform extraction of DNA with Ether instead of Chloroform, because the latter was not readily available in pharmacies where she came from whereas Ether was. I may have missed the outcome and will have to try and follow up on this one; solvent replacement is an interesting area that calls for some serious work.

It is my opinion and that of one Brian Degger that biotechnology is destined to become a benign science requiring few or no artificial or non-biodegradable chemicals. I don’t think we’re there yet, but I think we can transition that way through creative substitution and research into alternatives.

Plastic and Glassware – Have!

It’s easy to buy lab plasticware and glassware on ebay and aliexpress, so this isn’t an area that needs work. It does behove the DIYbioer or Garage Biotechnician to try and use glassware where possible, to cut down on expendable costs and pollution. The amount of plastic waste in the average lab today is pretty sickening, and disposal of biohazardous plastic is incompatible with recycling.

As mentioned above, most glassware can be sterilised by autoclaving in pressure cookers, and the larger pipettes and erlenmyers can be bleach/sterile water/alcohol treated to sterilise them, and then kept in a sterile bag until needed.

Enzymes – Don’t Have!

This is the biggie. Enzymes are expensive, prone to degradation, come with an expiry and may not even be legally available to you despite carrying virtually no hazards in the main. Companies selling enzymes often refuse to sell to individuals, which may be a choice of theirs or a legal requirement (as in Germany, I am told).

It falls to the community to solve this issue by focusing early efforts in established labs on producing easily purified enzymes for routine tasks; the key enzymes for biobrick assembly, a high-fidelity polymerase such as KOD, Ligase and Phosphatase.

Of course, it then falls to the community to also create buffers for each enzyme which can be easily created and distributed as concentrates to hobbyists worldwide. Time will tell how long this will take. Ultimately, some things cannot be reasonably produced by individuals or even the average garage lab; dNTPs and ATP are an example of something pretty far out of reach for DIYbio or Garage Biotech to produce in-house, so someone will have to apportion this from wholesale supply or mix up buffers for everyone.

Agarose, Sybr-Safe, Running Buffer, Loading Buffer – Sort of Have

Ok, so we can homebrew a loading buffer from glycerol and certain food dyes, so that’s not a priority. In fact, commercial loading buffers probably have too much salt for sodium-borate based gels, which are the most practical solution.

We can even replace Sybr-safe with methylene blue from aquarium stores, although the former is by far preferable for more accurate and sensitive results, and works really well with a Pearl gelbox (see above). You can probably buy Sybr-Safe easily enough, and because it requires no special disposal procedures it works out cheaper than the apparently less pricey ethydium bromide.

However, Agar just doesn’t work nearly as well as Agarose; the real-deal is pretty much necessary for trustworthy gel runs and predictable results. Unfortunately it’s pretty expensive, so it might be nice to try and make a transgenic bug that produces pure agarose, so that it can be homebrewed by hobbyists. For want of such a back-room DIY solution, agarose must be purchased. It can be had from ebay or from individuals on DIYbio who can buy in bulk and resell sachets.

The other difficulty is in procuring the running buffer that is used to make the gels themselves and then to run them in the rig. The cheapest option is to buy sodium borate and use that to make cheap running buffer, although watch out for racing ladders! Because a sodium borate gel is so low in salt relative to many ladders and loading buffers, the high-salt ladders can race through the gel and mix up results. Sodium borate is also known as borax or disodium tetraborate pentahydrate.

The other option is to buy some uber-expensive TAE or TBE buffer, though this is fraught with issues also. TBE can mess with downstream enzymatic reactions because of the larger amounts of borate, and the EDTA in both buffers is a highly damaging environmental pollutant. Both buffers also suffer issues with overheating and require lower voltages, so they take longer. The single upshot is that you can expect commercial ladders to run normally in them.

Perhaps the community would also benefit from a “ladder” strain of bacteria which carries a plasmid that can be cut several ways to produce a nice ladder of equal salt content to other samples, making the cheaper sodium borate option more useful?

Cultures

Labs strains, mutants and wild type bacteria are always welcome in a lab. It’s true that you can get buy with only what you have on hand, but the ideal lab workhorse is E.coli strain K12 because it is so excellently understood and studied. However, biosafety becomes an issue if you want to collect microbes.

Microbes are rated 1-5 on the Biosafety/Biohazard scale, where 1 means that they pose no threat to human, animal or crop, even the immunosupressed. Things get riskier as you go up the scale, and generally require licenses.

Interesting biosafety 1 microbes include Photobacterium phosphoreum, Gluconacetobacter xylinum, and Dienococcus radiodurans, all three of which are likely to be easily extracted from things you can procure without difficulty (respectively seafish, kombucha pancake-cultures or rotting fruit, and sun-baked dust). Finding these bacteria in your neighbourhood may require help or instruction from a protocol, which is discussed below as a general requirement in DIYbio.

For more “pedigree” strains, there is no reason that biohazard 1 strains can’t be shared in the post. Be sure to do this above-board; after securing your bacteria carefully within the packaging, label the outside clearly with the words “Non-hazardous biological sample”. Several of my colleagues at an academic lab have transferred cultures like this and have encountered no trouble, even when posting to the USA.

The idea that a community might retain a database of who and where to seek culture X was proposed on the DIYbio mailing list, and this “Cloud Cultures” idea may develop into a community effort as worldwide DIYbio labs with their own storage solutions start to become a reality.

Skills and Protocols

This area doesn’t require much breakdown. The area of DIYbio aims to spread knowledge and competence in Biotechnology to interested amateurs and professionals seeking more economical or practical means to practise their science. For this, we need to establish “DIY-able” protocols for a number of routine tasks, and also some interesting ones.

I personally enjoyed writing up a “how-to” of producing bacterial broth and isolating a fascinating, glow-in-the-dark bacteria with it. What surprised me however was how much interest it gathered when I presented it at Maker Faire, as did Brian Degger’s writeup and demonstration of DNA extraction using household ingredients. People like to see science, but people like even more the idea that they can do science and take part. It’s at the heart of DIYbio as a movement.

Engaging writeups of scientific procedures that are not only interesting but also practical and didactic will help get people involved, but to prevent them becoming unmoored and drifting on, we need a robust foundation of practical instructions on how to take things further and start doing real science. We need a “Biohacker’s Guide to Biohacking”.

Take Home Message

There are a few things above that stand out as immediate, achievable aims that should be met ASAP. It would be great to get a trace-heating Incubator board going, because that’s a mail-order solution to a common stumbling block. Likewise, if it isn’t difficult to dream up a spectrophotometer, DIYbio needs this soon too.

The other take-home is more positive; most of the above is immediately achievable. For less than €1000, you can probably buy all the equipment and consumables needed to start doing microbiology experiments. If it’s legal to do so where you live, you can probably also start buying the solvents, chemicals and enzymes needed for more in-depth molecular work.

Hell, Mrgene.com accept payment by credit card and deliver DNA plasmids in the post. Calcium chloride (readily available on ebay) can be used to render cells competent for transfection using a centrifuge, which can in turn be had for less than €70. You barely need any special equipment to make your first GMO nowadays, so why wait if you’ve had a great idea?

Posted via email from Cathal Garvey

A week or so ago I sat down in OpenSCAD and spent some time modelling something I've wanted to make since I got my Makerbot almost a year ago. For a while, I hadn't made it because OpenSCAD's compiled binaries didn't support extrusion, which I needed to create a twist-y backbone. For a while after that, I was exceedingly busy with marriage and mousetraps. I still am busy with mousetraps but we all need a holiday now and then!

So, when I successfully printed my first DNA molecule, I was happy with the results, but I knew (being a geneticist and perfectionist) that it was only a sculpture, and that the real thing wouldn't print correctly on my Makerbot. The angles were too steep in real DNA, so I settled for a sculpture of the real thing for then. And what success! Three people have since printed their own DNA molecules, as can be seen from the Thingiverse page, and their copies are even prettier than mine.

But, I wanted more, so I recompiled the model using parameters matching real DNA, and created the updated "B-DNA" model, which had the correct diameter-to-turn-length ratio, the correct number of bases per turn, and the correct angle of the bases (about -1 degree from horizontal!). This model I put on my Shapeways store, and ordered immediately in their standard plastic material to see if it would work.

Today, I got it! The picture is of the model I just received. It's smooth and sturdy, slightly flexible too (unlikely to break!). At last, a perfect little model of my favourite molecule, something I've always wanted! Better yet, someday I can order it in gold-plated steel when I've made my fortune on DNA.

For those who have a Makerbot or a RepRap, the DNA model is here for printing, editing and sharing. It's under a Creative Commons Attribution, Sharealike license, so you're free to copy, edit, share and even re-publish the model as long as you give proper credit for the original model to me, and share your own derivatives under the same license.

For those who want more, you can get both the "sculpted" DNA model and the accurate DNA model from Shapeways. Perfect presents for biologists, geeks and DIYbio enthusiasts.

Posted via email from Cathal Garvey

In essence, the idea is to make bacteria who "blink" as whole colonies, by creating and destroying a fluorescent protein in an on-off manner at the same time as a group.

This has been done in single cells, but as you might imagine the pattern of on-off gets lost in the crowd of a whole colony of millions, so the effect isn't clear to the naked eye. The importance of the research cannot be understated; if synthetic biology is going to be the next revolution in technology (and it is), it will need to be based on a platform of genetic systems that can be adapted to engineering goals. Having a system that can offer a timed, predictable output is essential.

Basically, the computer processor that drives the device you are using to read this is utterly dependent on a "clock", the speed of which is often used to sell the computer or device in question (New processor! 84GHz! Buy Now!). Biology has no such "clock" yet with which to synchronise the actions of engineered cells, and these papers suggest ways of doing so.

The problem is, both papers only offer mathematical models of their systems, even though they have fully detailed the genetic basis behind them. For the latter paper, I have calculated the cost of creating the engineered bacteria they describe using Gene Synthesis, based on the sizes of the proteins they describe in the paper plus 20% to cover regulatory regions and cloning sites necessary to construct the system in question. I assumed a cost of €0.30 per nucleotide of DNA, consistent with the current price from Mrgene.com.

So that's the three repressors, LacI, TetR and cI, plus the Lux proteins LuxI and LuxR. The links lead to their articles on Uniprot; the relevant datum is the number of amino acids, each of which corresponds to three nucleotides of required DNA. The added 20% is the number of nucleotides I expect would be added to cover restriction sites at either end of each ordered sequence, plus promoter sequences to make the researcher's job one of simple assembly.
The tally? Only €1347.

Now, that may be significant money to an individual, but frankly if I'd designed that system to an extent that I would be comfortable writing a paper about it, I'd feel stupid not to order the DNA and prove my hypothesis. To have the world's first independently synchronous bacterial clock, which sat there on a petri dish and blinked at me every few minutes? Not only would it be satisfying, it would be newsworthy and game changing. And it would add a ton of weight to the published result, rather than a slew of mathematically modelled predictions. In other words, it would be seriously awesome stuff for your career, a solid investment (better than a TV anyway!)

That's €1,347 of money that the authors of the latter paper (not to mention the first, which might have been cheaper) never spent.. but why? Because testing their hypothesis could have disproven it? Even a negative result adds to the body of knowledge in such a young field.

I'm frankly pretty amazed. In my research on oscillators, I've seen a few but none yet that have used quorum sensing to synchronise cells, even though two papers have been written on the topic with favourable predictions of worth. Considering the cost (compare this to the cost of the Craig Venter Institute's basic copying of an existing bacterial genome, a staggeringly expensive task which ultimately added little to the body of knowledge on the topic), this lack of follow through is confusing. Especially since this is clearly the field of choice for the authors: some of their publications since that paper have been equally exciting.

If they're not going to do it, I will. As soon as I have a lab capable of creating transgenic bugs and enough money to throw at it, I'll feel compelled to make the world's first blinking bug. Who knew there was such low-hanging fruit awaiting a fresh synthetic biologist? Anyone feel like helping me bootstrap ASAP, and getting a free blinking bug as a prize?

Posted via email from Cathal Garvey

I know this makes me a bad person, but my first thought on seeing this clip was: "Explosive… dance routines?!"
I can do this for "bioterror agent" too. "Yea, I've seen some pretty.. sick moves!"

Clearly I watched too much CSI: Miami in my TV-watching days.

Posted via email from Cathal Garvey

As fully outlined here on Derek Lowe’s blog “In the Pipeline”, Quackwatch is getting sued by some quacks because he highlighted their use of a misleading diagnostic and how it could be used to push unnecessary and potentially dangerous treatments on clients.

Quackwatch have a Paypal donation system, so if you feel like committing a fiver to their defense fund, I’m sure it’d be much appreciated. After all, I’m guessing there’s more money in quackery than in exposing quackery!

Posted via email from Cathal Garvey

I’m a fool to be up this late, but I have to give a quick shout out in favour of the Biocurious Hackerspace, and a strong suggestion that you help them reach their goal on Kickstarter.

Biocurious is planned to be a place where people can learn about and “hack” biology with access to proper, modern biological lab equipment and with close communication with fellow enthusiasts and fully trained biologists to help out. The projects they have mentioned doing include bioprospecting for new species in the San Francisco bay, genotyping blood samples to give an estimate of cancer resilience, art-prints of people’s cells and more.

Go and donate something, they could really use the money if they are to get their goals met, and the people behind this will make it happen if they raise enough funding.

Posted via email from Cathal Garvey

Just for love, I designed and printed myself a little sculpture of a DNA double-helix on a small base. It’s going to be my desk ornament in the lab for the next few months while I try to finish my project on time and write a thesis.

Of course, nowadays if you’ve made something you’re proud of, it’s a trivial thing to offer it for sale to others with similar nerdy interests! And so, I put this on Shapeways.

If you’re not familiar with Shapeways, they will print out your 3D files in virtually any material, this includes plastics, glass, steel and gold-plated steel. It’s crazy but brilliant. And items can be offered for sale somewhat like Lulu does for books.

So if you feel like a premium, gold-plated model of DNA on your desk, you can hop on over to Shapeways to order your DNA Sculpture!

Perhaps if this sells well it’ll help cover the costs of all the quite excellent stuff I’m buying from Pearl Biotech. Even if they are worth every penny.

Posted via email from Cathal Garvey

Ask any RPG gamer whether they buy their books locally anymore, and you will usually get one of two answers. Either they don’t, because it’s so much cheaper to buy them online, or they do, and they do it to support their local gaming store.

When Gaming Retail Becomes Charity

But, when did it become a matter of charity whether or not to buy locally? The fact of the matter is, for a variety of reasons tied into Gaming Culture and the profit margins of selling books of any sort, it’s simply not cost-effective to run a traditional RPG store anymore.

To run any Brick-And-Mortar store, you must balance the cost of rent per square meter of floorspace against the profits generated by that same floorspace due to customers buying your products. This is a pretty fundamental principal of retail, and you can see it in action everywhere you look. For example clothes shops must weigh the benefits of stuffing as much stock as they can against the discomfort it may cause and against the price of their clothing. So, very up-market clothes shops with huge markups on their clothes can afford to make shopping feel like a “premium experience” by leaving lots of floorspace, whereas Pennys and TK Marks try to stuff as much clothing into as little space as possible while still allowing customers to get in and buy things.

For gaming stores, the problem is that much of their stock sells slowly and carries an unexceptional margin, making it a business with underwhelming profit margin generally. Of course there are exceptions, but this is certainly true of the RPG book section. However, internet retailers do not have to pay nearly as much rent per square foot, as they can simply stack the books up high and not leave room for customers. Perhaps they don’t even pay commercial rent, as they can work out of home. Because of their drastically reduced outgoings, they can afford to charge far less than Brick and Mortar shops, which attracts buyers away from the latter. Which of course, feeds the cycle; to justify keeping an RPG book section, local shops must hike the margins to balance their poor sales against the floor area they occupy.

Eventually, the battle is lost. Shops either continue to stock RPG books simply because they attract clients without any expectation of meaningful profit, or they stop stocking the books altogether and focus on items with better margins; Card games and Warhammer for the most part.

Compounding all of this is the fact that the local gaming shop typically becomes a social hub for the local gaming community. This may seem like a boon, but in order to make people feel welcome you need to provide some floorspace in which they can socialise, floorspace that is not generating any profit. Asking players to “support” your shop by buying products they usually get cheaper online is an artificial sort of business, and is not sustainable. Players come for socialising more often than for books; instead of spending their floorspace stocking books, gaming stores ought to be enabling and monetising the social element of gaming.

Publishers Collapsing

It would be a closed case and just another story of gaming evolution if things were still going strong for games publishers. However, book publishing ain’t what it used to be, and even some of the big RPG publishers are dying. Between technological innovations that undercut their existing business model (make and sell books), a lack of spending power in their target audience (think: The “R” word) and the buy-one-and-share-with-friends nature of RPGs, things aren’t working out. E-publishing is growing quickly as a way to sell books without much cost, and the slew of smaller-scale e-publications from White Wolf are a good example of emerging coping strategies in relatively savvy companies.

The problem is, although gamers will certainly buy these e-books for reference texts, they still want to have physical copies for the mainstay books of the games they play. Having to print fewer books to satisfy their customers may seem like a tonic for the publishers’ outgoings, but the deficit isn’t wholly covered by the rise in ebook sales. What is needed is a way to have the cake and eat it, to provide books while not having to make, ship and sell them.

On-Demand Publishing

One potential avenue which fulfils the have-cake-and-eat-it desire is to employ on-demand models for publishing of books. Avid White Wolf fans may remember that several of their titles were published through Lulu.com in this way, including a cheaper soft-bound copy of Exalted 2e Abyssals and some nicely sized softbacks for LARP World of Darkness. For reasons I cannot understand, White Wolf pulled their collection from Lulu. This didn’t make any sense to me, because by Lulu.com’s nature, it is a zero-risk model for book authors and small publishing houses; if books don’t sell, you lose nothing. If they sell, you get a profit. Win-win?

However much I would have rathered they maintain their collection at Lulu, it is not the answer to their woes anyway: The lead-times on getting a new release would exceed the time taken for it to come in stock elsewhere. Nor is it a feasible solution for Brick and Mortar shops, who are ultimately agnostic about methods of production, making orders from Lulu a time consuming and pointless change for resellers. Lulu would make the perfect back-ordering system for people who missed the initial run, or for out-of-print previous editions, however.

Far more exciting is the advent of point-of-sale book printing, such as the Espresso Book Machine. This machine can print a 300 page book in 3 minutes, at a cost of about €3 for materials. Books are “perfect” bound with colour covers and black and white interior. In addition to a vast archive of out-of-print and public domain books that can be bought by interested customers, the company behind the machine has licensing deals with several major publishers, including a wing of Ingram that deals solely with alternative publishing. Even better, the interface on the machines can be used by customers to contribute their own work for sale on any other machine worldwide, making them an interesting new means of distributed publishing for unestablished authors.

Imagine a world where local gaming stores have adopted such machines. Rather than spending floorspace on a (invariably poor) selection of RPG books, they have a single machine capable of fulfilling the needs of even the nichey-est customer, as well as mainstream customers looking for regular or out of print books. Because of the low cost of local printing for smaller publishing runs (such as RPGs in general), lower prices can start to entice customers back from the internet with the promise of a book now instead of in several days. Publishers can start to publish directly to these on-demand sites, making the dissemination of new books an instantaneous task requiring no more effort than uploading a manuscript. They no longer have to pay for manufacturing or distribution, leaving that to the automated machines in the gaming stores.

In fact, this distributed as-you-desire-it model allows Errata to be amended into the core manuscript so all future versions of a book have the correct text and ruleset, an advantage wholly lacking from the limited print runs the publishers depend on at present. For some games, this becomes a serious headache: Exalted Second Edition, for example, has hosts of errors derived from the simultaneous writing of the core ruleset and an expansion based upon it during the launch of the new game, and the errata for these books is distributed across the publishing set. Referencing charm revisions is annoying and creates arguments among players; there would be a great motivation to upgrade to a “fixed” manuscript, should one become available at the local store.

The fact that these printers spit out black-and-white books that fit a certain build envelope may seem limited to some enthusiasts who want full-colour, stitched-binding “trophy” books for their shelf collection. This actually opens the door to a “Premium” business route for games publishers through Lulu, where they can stipulate far more about the quality and content in the books and thus offer a more attractive product for those willing to pay extra.

This solution could work now, except for OnDemandBooks charging upwards of $75,000 for their machine. Clearly they don’t realise the immediate need for a solution to the Publishing Industry’s immanent death. If this machine could be brought down to €5,000 and sold globally (it is currently limited to the US, UK and Australia), then publishers (RPG and otherwise) could strongarm or even subsidise point-of-sale shops into installing them. For that matter, if it could be leased to shops by Publishers, it could achieve a similarly dramatic effect even in small, struggling businesses that otherwise couldn’t afford the buy-in.

But, there is no sign of this happening just yet. Hopefully ODB will realise they are sitting on a goldmine and start to experiment. If they wanted a niche market to test this potential on, they couldn’t do better than RPGs. If they don’t carpe diem, someone else will make a book printer and steal the market from under them anyway. Maybe the competitor would even care to release their designs into the public domain?

Tablet Computers

As an alternative to On Demand Publishing (and probably an inevitable successor of in any case), the rise of usefully interactive book reading systems is about to go mainstream. I say “Usefully” to differentiate from devices like the Kindle or Sony Book Reader, which are explicitly designed for Novels, and deal very poorly with richer, picture-riddled RPG books and make page-searching an act of torture.

No, for RPG books that may need to be constantly re-opened on different pages, you need something more powerful, something like a Tablet Computer. Despite the ebook-related hype surrounding the iPad, it is a poor choice of tablet and has profoundly limited capabilities compared to virtually every competitor, not the least of which is the lack of extended storage (my collection of Exalted books alone is 1GB). The lack of USB ports or any other means of convenient connectivity also means it will be a chore to actually load books on and off the device, or to carry libraries of reference texts in a USB key ready for use. RPG publishing would do well to skip Apple’s early-market attempt at a tablet and take the longer-term view, particularly given the walled-garden, censored nature of Apple’s content stores.

The real game-changer about to enter this market (it is hoped, because it could be vapourware) is the Notion Ink Adam. For gaming, you won’t be able to do better. The device has a dual core processor with a separate graphics processor, meaning rich-content books will be trivial to read in real time. The battery life is superior to most competitors thanks to the processors and the display, which can be switched to an ultra-low-power reflective mode (resembling motion-capable e-ink) to make reading easier. The design is ergonomic, and the device has 3 USB ports for complete connectivity with external storage or whatever doodads you feel like using. Best of all, it will handle Flash 10, meaning embedded content in PDFs will work as expected, and Notion Ink promise an innovative e-reading system that may prove to be superior to the traditional on-screen PDF experience.

If the Adam fails to materialise, it doesn’t matter really. Someone else will make the Jesus-Tablet and kickstart the market. Flashing forward ten years, tablets like the Adam could be commonplace, having replaced the laptop for convenience and perhaps partnering with the mobile phone as a common communication system. If every gamer can reasonably be expected to own a capable tablet on par or superior to the Adam, why even use books anymore? PDF cannot compete with books at present due to the punitive lack of convenience, but this factor is already diminishing and may wink out entirely with the advent of touch-controlled book readers. It is likely that EPUB will replace PDF, or a new format with more interfacing flexibility will take over, but the light tablet form factor is an easy match for a library of reference texts.

It is even conceivable that publishers will start selling their “Books” as applications, containing a reference text and a convenient game-management system or even teleconferencing suite built in, which could transform the way that games are played. But, such ideas are getting ahead of the point of this essay.

Future of Gaming Stores: The Transition from Incidental to Deliberate

Social Hub Where does this leave gaming stores?

In one possible outcome, the publishing system changes to put reasonable distribution back in their control, but they are now left with extra floorspace and the demand for books is really just returning to its prior “business as usual” level; that is, unspectacular. It is an improvement, but not the complete solution to the problem of gaming stores making too little money; this is a problem far more complex than just RPG sales.

In the latter outcome, which might well follow naturally even if On-demand publishing takes off, Gaming stores are entirely divorced from distribution and sales of Gaming Titles. In reality, this doesn’t really affect their bottom line much. However, the only reason most Gaming Stores bother with RPG books is because they ought to maintain an inventory in order to attract customers into discuss gaming and keep the hobby vibrant. If a grocery store doesn’t stock milk, you won’t think of that store first when you need to get bread, because you’re used to going elsewhere. So if gaming stores stop selling the things Gamers enjoy, there is less attraction for them in a shop and the shop loses a great deal of incidental custom.

The solution may be for Gaming Stores to look at what gamers are coming for and what they want: society. Gamers like to have a place to come and meet others, talk and enthuse about stuff, try out new games, plan and play games or tournaments, and sometimes even seek employment relating to their hobby. Gaming stores provide this as an incidental thing, but they could start to deliberately support and enable gaming society.

Already some stores rent prepared and well-stocked rooms for running card games, war-games or Roleplaying Games, charging for access by the hour. For gamers accustomed to playing at home, this might not be a big lure, but an erstwhile group may lack a convenient location. By offering “premium” features, like Livegame Recording, media systems for ambient music or mood lighting, reliable teleconferencing for remote players and a ready supply of snacks, tea and coffee, a gaming store could attract groups to rent rooms for easily as much as €5 each per hour, which may be more than they are earning for that floorspace already.

Additional methods of providing an atmosphere of society might involve: * Creative use of media effects, like the projection of real-time gaming news or tweets on a wall to provide discussion-fodder. * Screening of gaming-related movies in the event rooms, somewhat like a niche-cinema. * Maintenance of a local gaming ledger into which gamers can write anecdotes to share with others for fun. * Free Ads for gaming and a place for young authors to showcase their work. * Workshops for Model-Making, games-writing and strategy; capitalise on the playing of games rather than on their sale. * Offer “trial days” for people to test out the games that interest them but which they don’t want to risk buying. * Award achievements in games to encourage a friendly competitive effect; players earn medals for killing 10 opponents in one conflict, or dying spectacularly. Make the shop a central, entertaining arbiter of gaming attainments.

Some of these can be directly monetised, others can’t. Ultimately shops are about making money, and one which fails to do so will fold, social hub or no. But in order to remain viable, shops must adapt to changing needs as they occur, and needs are changing now among gamers. A shop whose response is to post “Buy local, support us!” is shortsighted and doomed.

Last Word: Publishing in Microcosm

Although the Gaming niche is particularly social, this transitional scenario we are seeing mirrors what is happening to publishing in general, albiet more slowly. I have read it suggested that publishers no longer sell books in order to directly make profit from book sales, but rather to try to provoke the next big blockbuster to which they will have movie rights. This wouldn’t surprise me.

The larger publishers have more momentum behind them, and large companies take longer to fall. See Microsoft, Sony and Motorola for examples, and see how the latter is making a comeback due to their adoption and enthusiastic backing of a new and adaptive technology (Android, in that case). Those publishers that adapt will survive, while those that try to rebrand and push traditional book sales will probably not.

Because Gaming is a small niche, and because these problems are already beginning to manifest at ground level (Closing games stores, a rapidly shifting precedence to online publishing), it could end up being a model for the larger industries who need to see an example of a working transition to follow themselves. Achieving this transition will require dialogue and co-operation between the point-of-sale (and hobby-fostering) stores and the publishers, so that measures to replace the old business schematic can happen in short order with minimal fuss. If machines like the EBM are going to be needed to make this happen, then OnDemandPublishing.com need to be clued in fast, so they can tailor their pricing and distribution to match this testbed market, and perhaps help traditional publishers buy into their product more readily.

If Gaming sales become a social endeavor, expect to see this happening in smaller bookstores also. Innovative ways to get people interested in reading and building loyalty to a local bookstore may lead to a renaissance in reading-for-reading’s-sake, which would be good for everyone concerned. The “One Book One Twitter” concept is a great example of such innovative social projects for books and literature, and I’m sorry I missed the boat when they started reading American Gods.

It’ll be an exciting decade in Gaming. Expect to see casualties, and expect to see changes in your own backyard. These could be positive, bizarre or disappointing, but don’t be afraid to offer suggestions to your local store if you think of ways for them to tailor their service to your needs as a customer. After all, that’s what they’re there for!

Note: As with all things, I write, this is released under a Creative Commons Attribution, Sharealike License 3.0 Unported.

Posted via email from Cathal Garvey