Re: [DIYbio] Re: IndieBB Campaign Survey; Help Appreciated

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Hi Mike!
Thanks for the feedback; reading through your email I think I recall
some of those queries from the survey, too.
Given that feedback (from you and others) I am currently writing a
Technical FAQ, which will allow me to offload the "hard stuff" from the
crowdfunding page while not doing you and others a disservice. I do want
this to be as open and informed as possible, without scaring newcomers
with excess data.

Anyway, while I write the FAQ, here are some quick answers to your
questions; I'm glad you asked!

> --I'm a grad student, and get to do the bio I'm really interested in
> at "work." So I would only use IndieBB myself if it offers something
> I can't get on Addgene etc.

Well, if I'm honest you may be able to find the Colicin V cluster on
Addgene, already; it's been thoroughly studied. And if you put work into
it, you could assemble that yourself with a fluorescent protein and a
stable origin of replication, add a multiple-cloning site, and you'd
have something that looks like IndieBB.

Two advantages to IndieBB: I'll be testing, modularising and optimising
(if appropriate) the Colicin cluster for you, so you don't have to do
all that hard work, and it'll be available to others; addgene is only
available to the Ivory Tower.

> --However, I'm very supportive of DIYbio and educational tools being
> available to public. I'd be willing to fund just for that reason.

Thanks! "Educational Kit" is the primary goal here, with "free/libre
biotech platform" as the close second. I think the more people who
handle biotechnology and feel a sense of ownership and self-control over
it, the less biophobia we'll see in the public sphere.

> --What worries me is the unproven non-antibiotic selection. If it was
> easy, we'd already be seeing more of it...

I don't think so, really. My experience of academia was that the
academic environment (not personality) forces people to accept tools
that work "well enough" because they are not funded to improve
fundamental methods. "Publish or Perish" means that you have to always
be working on speculative, publishable stuff, and that means you have
little time to be experimenting with better methods.

I recall suggesting an alternative transformation procedure to my
labmates (the procedure that'll be in IndieBB; the "TSS" method) once.
It was striking; one group made their own CaCl samples, the other bought
in Top10 cells at €20/sample. Neither were interested in TSS, because A)
being mid-experiment, they had to stick to consistent methods and B)
they had no time to "waste" on even time-saving experimental methods.

I know that's not universal, of course, but when antibiotics are
provided cheaply to university labs, it's not a big burden to just use
them and get on with research. It'd be nice not to have to use
antibiotics, but it's not worth three to six months of work, which is
what I'm devoting to IndieBB!

Especially since, as you note and I never denied, biotech is uncertain;
this mightn't work, even though I'm fairly sure it will.

> --I've witnessed someone i respect getting their kickstarter funded,
> spending the $, and ultimately not being able to deliver...it ain't
> pretty. Don't get me wrong, you seem to have a lot of plasmid/gene
> hacking knowledge (definitely more than me!)

I think this is implied in all crowdfunding projects though, and it's
not exclusive to those with a technical tendency to not-work (as biotech
has). I've backed books that never got written, for example.

I've budgeted accordingly, and it's part of the high price tag. It's an
all-or-nothing campaign and I've budgeted for three prototypes; why?
Because in my experience, you need at least two to test if something
really works. The third is for the finished product.

If it fails, then funders will have my sincere apology, but that's
science/engineering for you, and I'll never promise that this will work
until it does.

You say I have more plasmid-hacking knowledge; perhaps. I've made a
plasmid before that didn't work (fully). But, it did work partially, and
if I had 16,000 to make another two prototypes, I think I could have
made it work fully. That was far more speculative; in my *opinion* (no
guarantees), IndieBB is a much more down-to-earth bet than my prior work.

> --Link to the blog post with details sounds great!
> --Some endorsements from respected biologists (DIY or academic) would
> be nice...this is one thing that made me feel good about Glowing Plant

It's my curse that I work alone; not by choice, but by simple isolation.
So, while I know a great many people in this field, none of them are in
a position to join the project full-time.

I can offer citations for Colicin V, of course; in its natural context,
it is a self-selection system. However, in the high-growth-rate
environment of E.coli at 30C*, perhaps it'll need improvement; I'd err
towards putting Cvi and CvaC (immunity and toxin proteins, respective)
under a stronger promoter and keeping the transporters CvaA/B the same.
These sort of improvements have no precedent, because nobody's looked at
using Colicin V as an engineering tool in this manner, so I can't offer
citations or testimony towards them.

Hope that answers a few questions. I'm still writing the FAQ: If anyone
else wants to have a *technical* question answered, post it here and
I'll include it!

As to "regular" FAQ, of course I welcome that too so I can better
structure the main pitch.

Best,
Cathal

*Yep, 30C: Being forced to use wild-type GFP imposes this.

On 19/02/14 16:30, Mike Horwath wrote:
> Hi Cathal,
> I responded to your survey earlier. I'm probably one of the people you can
> lump into "didn't contribute due to wanting more information".
> For the sake of friendly discussion,
>
> --I'm a grad student, and get to do the bio I'm really interested in at
> "work." So I would only use IndieBB myself if it offers something I can't
> get on Addgene etc.
> --However, I'm very supportive of DIYbio and educational tools being
> available to public. I'd be willing to fund just for that reason.
> --What worries me is the unproven non-antibiotic selection. If it was easy,
> we'd already be seeing more of it...
> --I've witnessed someone i respect getting their kickstarter funded,
> spending the $, and ultimately not being able to deliver...it ain't pretty
> --Don't get me wrong, you seem to have a lot of plasmid/gene hacking
> knowledge (definitely more than me!)
> --Link to the blog post with details sounds great!
> --Some endorsements from respected biologists (DIY or academic) would be
> nice...this is one thing that made me feel good about Glowing Plant
>
> Mike
>
>
>

--
Please help support my crowdfunding campaign, IndieBB: Currently at
25.4% of funding goal, with 23 days left:
http://igg.me/at/yourfirstgmo/x/4252296
T: @onetruecathal, @IndieBBDNA
P: +3538763663185
W: http://indiebiotech.com