As I am redesigning the main pitch-page for IndieBB in order to appeal more to the currently-untechnical audience, what little technical detail I've reserved for the main page will have to be stripped out; I'm taking this opportunity to write up a “Frequently Asked Questions” post to address the more technical queries I'm seeing in the survey, tweets, comments and other correspondence.
I will soon also post a less technical FAQ, but given that “Less Technical FAQ” is now the design goal for the main IndieBB crowdfunding page, I'm considering it a lower priority.
I can edit and add to this as newer technical questions emerge; please make such queries on the IndieGoGo “comments” pane if possible so other users can see the question and the response!
To the really dedicated techies, I'll try to return to this with my citations and literature where useful.
Q: What does IndieBB Stand For?
“Indie Biotech Backbone”. Backbone is used interchangeably with “vector” as a term for a plasmid that's been designed for industrial or research use. Because this is dedicated to Indie Biotechnologists, it's named appropriately.
Q: Technically Speaking, What's In IndieBB?
The pitch for “beginners” is that IndieBB makes bacteria fluorescent, but there's a lot more going on under the hood. To make IndieBB as easy as possible, it includes a novel self-selection system that (if it works as intended) removes the need to include or require antibiotics in the kit. Because this is the most novel element of IndieBB, it's described further below.
IndieBB will also include a gene for a fluorescent protein called “Green Fluorescent Protein”. For beginners, this is the part they'll actually notice; from a technical standpoint, it's actually the least interesting part! Because IndieBB must be a Free-as-in-freedom product, it cannot consist of patented parts. Sadly, in this degenerate age, patents on substantially natural genes are permissible and long outlast their novelty or commercial use, so even very old variants of GFP like the original “enhanced GFP” are still patented. GFP itself was patented, insultingly enough, despite being entirely natural, but fortunately the patent has since lapsed and it can be used as-is.
One noteworthy consequence of sticking to patent-free “natural” GFP is that it comes with all the drawbacks of that protein, now alien to the coddled emerging generation of synthetic biologists. I've accounted for the worst of these, the low fluorescence, by using codon optimisation software of my own design to make sure that plenty of the GFP is produced. But one significant drawback remains; it can only fold at ~30C, so cells can't be cultured at the more usual 37C.
Finally, IndieBB will contain a multiple-cloning site and an origin of replication. Neither are yet set in stone, but I'd like the MCS to be more generally useful than simply for routine cloning; it'll be biobrick compatible, for starters, and I'd like to offer defined, easily-primed regions (as in, PCR primer landing sites) for use in techniques like gibson assembly; not essential, but maybe useful for some.
At the suggestion of an early supporter, I will also aim to include comparably easy-to-prime sites surrounding the submodules of the plasmid.
Q: Tell Me More About The Self-Selection System
Selection is the process of removing E.coli from the mix who have not absorbed the DNA you offered them, and the ongoing process of preventing cells from losing the DNA afterwards. It is normally accomplished by providing DNA that makes cells immune to Antibiotic, and then applying antibiotics to the culture so that only cells with the DNA survive.
As getting antibiotics for selection is otherwise a large stepping stone for beginners and amateurs to overcome, I'm hoping sidestepping it will facilitate more people self-educating in biotechnology.
Specifically, the system to be used is based on the “Colicin V” (AKA “Microcin V”) system found naturally on some E.coli plasmids in the wild. Colicin V is a tiny protein that is toxic to E.coli (and only E.coli), but cells that produce the toxin are themselves immune to its effects. This means that, by copying the natural state of Colicin V, we can use it instead of antibiotics as a self-powered selection system.
Q: Will this (antibiotic-free self-selection) work?
Nothing can be guaranteed. However, I feel confident it will, because it works in its natural context, and behaves predictably in the many in-vitro studies of the system (Colicin V) that have been conducted.
It's true that the Colicin V system has never been deliberately used as a self-selection system (to my knowledge); if it had, then IndieBB wouldn't be as novel but at least I could offer some assurance of success. If it works, it's actually publication-worthy (open access only, don't worry) in a methods/protocols journal, I think.
Q: Why are you asking for funding for 3 prototypes?
Synthetic Biology is very error prone! In my experience, you need to behave like a Scientist for your first prototype, wherein you learn about the modules and how they behave separately. Once you understand them a bit better, you can start to think like an Engineer and tweak and recombine the modules to achieve the final result.
Specifically, the Colicin V system is best studied first as the “natural” sequence, and I plan to do so using brightly fluorescent, but not open-source (:() proteins so I can better understand it. Once Colicin V has been tested in its “natural” context, and I can say whether it'll work as-is or need “enhancement”, the second prototype is the same, with an optimised, further modularised, or otherwise altered Colicin V gene cluster, still using enhanced marker proteins to assist in testing. The final prototype is to be the final product; the optimised Colicin V system plus a patent-free fluorescent gene (optimised wild-type GFP is the current plan) which allows it to be considered “Free/Libre".
Q: Why are you paying yourself €3000 for 3 months’ work?
It's unfortunate that this was even asked.
One hopes, firstly, that the questioner is aware that this is far below minimum wage in Ireland. It may come as a surprise, and I am aware I appear much younger than I really am, but I have a family to help support! I might survive on less per month if I lived with my parents and ate noodles all the time, but I have a mortgage to pay for, kids to feed and clothe, and must still contribute to our costs.
For all this, I'm passionate about synthetic biology, and moreso I'm passionate about helping to create an open-source revolution in biotechnology. Sadly, nobody out there is yet offering careers in open source biotechnology, and so I have no recourse but to do it by myself as a full-time job.
€1000/month means that I can justify giving this my full-time work to make it happen. Without that money, I literally would run out of money and would have to seek alternative employment, relegating IndieBB to a weekend project, spared only the time between parenting, work and the necessities of continued survival.
I can guarantee the questioner that any project of comparable complexity that doesn't include “living wage” in the cost plan is just hiding it among the other costs. One cannot survive upon goodwill alone.
Q: How Will You Handle “Open Source” Development?
At this time, I'm planning to maintain an open repository on a platform that is most suited to DNA. It will be open to access, copying, editing etc., and I will welcome suggestions on the design. However, I'll be the BDFL on this project, and I don't promise to accept suggestions if they're uneconomical, feature-bloating or I just don't like them. You're always welcome to synthesise your own projects!
Q: How Will You Handle Open Source Licensing
This is very different, and non-trivial, compared to software, which can be covered by Copyright and those rights then inverted to provide irrevocable freedoms to users. There are strong parallels between the way that code, when compiled, can inherit the copyrights of its source-code, and how one might likewise allow DNA to inherit the copyrights of its “source”. And yet, this is not established or accepted as the norm anywhere that I'm aware of.
I have no intention of patenting the code to apply an irrevocable license, because I don't honestly believe patents can be used for good.
The Biobrick Public Agreement is a tempting option, but on its own it's more like the original BSD. It places (very minor) restrictions on how you package materials, and it eschews copyleft. I'm considering making minor alterations to the BPA to suit my needs, but it's possible I'll just use the BPA as-is, given my uncertainty as to how to enforce “Free/Libre” in DNA, anyway.
It might amuse readers with a Free/Libre background to know that I did ask Richard Stallman of the GNU Project and Free Software Foundation to offer some advice on the subject, but didn't get far!