IndieBB Crowdfunding Campaign: Help me make a great beginner's kit for DIYbio/synbio!

[Cathal Garvey]

Hey all,
As anyone on this list for more than a month can attest, our most common
FAQ here is "How do I get started?". More often than not, it's more
specifically "How do I get started in synthetic biology?", and our
answers can get a bit woolly.

The truth is that our options for beginners all suck. Most suppliers are
hostile towards independent buyers, so we tell new people to buy the
"refill kit" from Carolina and pretend to be a school, or we mail
plasmids of dubious provenance to one another. While that's good for
community spirit, it says a lot that we celebrate knowing the
approximate sequence of one of them at last.

Finally, we've talked a lot about the issue of getting, using and
disposing of antibiotics for this purpose a lot, and we've talked more
and more recently about removing the need for antibiotics altogether.

That's what I aim to do, and I'd really appreciate your help and support
making it happen. Here's my IndieGoGo campaign, freshly pressed:
http://www.indiegogo.com/projects/indiebb-your-first-gmo

The kit is designed for beginners, and for teachers and education
groups, to introduce people to the methods of E.coli engineering. It's
also designed to be hackable and to be fairly licensed in an Open Source
way that preserves the user's freedoms going forward. It's fairly
priced, and designed by and for DIYbioers. It'll be fluorescent, and it
won't need antibiotics.

If you're interested, please help me out and support the IndieGoGo
campaign. It's an all-or-nothing campaign, so if I don't hit the goal,
nothing is raised. If you know a bio/hacker, educator or student who'd
be interested, please let them know, too. And if you're on Twitter or
(ugh) Facebook, a shoutout to let others know would be really, really
appreciated. :)

For the purposes of fundraising and separating my usual noise from the
campaign, I've started a new twitter account for this, too: @IndieBBDNA

Gratefully yours, and looking forward to collaborating on this and
making it real!
Cathal
PS: As I know too well, nothing can be guaranteed to work in Synbio at
this point, so bear that in mind. However, this is the most conservative
design I've yet embarked on, and I'm confident it will work. So, bear
that in mind too. :)

[Koeng]

"IndieBB is further designed to do most of the work normally left up to you, most importantly the process of antibiotic selection"

Can we have any information on the self selection method? :D

Also I sent the page to everyone i think would be interested. Great job with the kit!

-Koeng

[Tom Hodder]

On 22 January 2014 20:01, Cathal Garvey <cathal...@cathalgarvey.me> wrote:

The kit is designed for beginners, and for teachers and education
groups, to introduce people to the methods of E.coli engineering.

HAHA. I might have indiegogo kit number 1, because I just bumped it from 25EUROS to 85EUR....

 

[Ricardy Ricot]

Sounds awesome

On Wednesday, January 22, 2014 12:01:00 PM UTC-8, Cathal Garvey wrote:

[Alex]

Looks great, Cathal! Wish I had the capital to grab a kit! I'll definitely send it around though!

Alex

[Cathal Garvey (Phone)]

Thanks Tom! Saw your contribution and I'm really grateful! :)

--
Sent from my Android device with K-9 Mail. Please excuse my brevity.

[Cathal Garvey (Phone)]

Of course! Full details will be forthcoming as the project develops and it'll be fully open source, though I'm gonna keep most of the juicy design details for supporters at first: it's part of the first perk, after all. :)

[Cathal Garvey (Phone)]

Thanks Alex! Well, every little helps and pledges can always be upgraded, but definitely what I need most right now is exposure. So thank you!

[Cathal Garvey]

This is very worth clarifying.
This kit will consist of DNA *and* class-1, lab-strain E.coli. Not DNA
*in* E.coli, therefore not a transgenic organism.

So, this kit ought to be legal to purchase nearly anywhere (yes, that
includes the EU), although actually completing the kit may not be legal
without a licensed premises to work in/on. As always, check local laws
etcetera.

On 22/01/14 21:59, Victor Helmre wrote:
> Hi Cathal! Really cool project, just what I've been looking for. Can I ship
> it to my home address in London, or is it better to send it to the London
> Biohackspace? Also, what hardware and software is included, and what extra
> equipment do I need?:)
>
> Cheers,
> Vic
>
> On Wednesday, 22 January 2014, Cathal Garvey <cathal...@cathalgarvey.me>

[Patrik D'haeseleer]

Auxotrophic strain? C'mon - you can give us a little hint at least...

Funded!

Patrik

[Cathal Garvey]

Thanks Patrik!

The details aren't gonna be NDA'd, but I am gonna parcel them out to
supporters first. ;) They can percolate outwards from there if ye like,
I just want to be honest about the "inside perspective" part of the
lowest-tier perk.

I will debunk myths about auxotrophy though before they snowball! No,
this won't require auxotrophy or specialised media or strains, it should
work in most E.coli strains. It's bacteriocin-based.

On 22/01/14 23:48, Patrik D'haeseleer wrote:
> Auxotrophic strain? C'mon - you can give us a little hint at least...
>
> Funded!
>
> Patrik
>
> On Wednesday, January 22, 2014 1:48:31 PM UTC-8, Cathal Garvey wrote:
>>
>> Of course! Full details will be forthcoming as the project develops and
>> it'll be fully open source, though I'm gonna keep most of the juicy design
>> details for supporters at first: it's part of the first perk, after all. :)
>>

[Koeng]

Totally buying this just for the operational bacteriocin genes xD.

I'll put the sacB gene from Bacillus with it (negative selection in presence of sucrose), to make a plasmid which you can clone DNA without selection and without electrophoresis :)

-Koeng

[Cathal Garvey]

:D Congratulations, I think you're the first person to speculate about
what you'd do with the plasmid! I'll have to think of a prize. ;)

[Patrik D'haeseleer]

Ah - now I remember!

For the uninitiated, here's an earlier post by Cathal on using bacteriocins:

https://groups.google.com/d/msg/diybio/_yCzEK8P6Sk/jCGdYDJeM74J

"Much better to focus on antibiotics that are too impractical to use in
humans anyway; bacteriocins look like a great option here. They are
protein-based, so they can't be taken as a tablet and they stimulate too
much immunity to be injected, so most of them are entirely useless for
human therapy. However, they can be pretty lethal against the specific
species and strains they affect. Further, the mechanisms of resistance
to these antibiotics are often evasive rather than degradative.

That is, while bacteria destroy ampicillin, allowing non-transformed or
plasmid-loss cells to survive alongside them, most bacteriocin
resistance systems merely protect individual cells against the
bacteriocin, without destroying it. This allows for longer culturing
times for transformed cells before plasmid loss becomes an issue, and
might even protect cultures from late-growth contamination.

With bacteriocins, you could even have your transformed cells *make* the
antibiotic, leaving the job of killing untransformed cells to the
transformed cells. That reduces your necessary ingredients from three
(bacteria, DNA, antibiotic) to two: bacteria, and DNA."

Patrik

[Cathal Garvey]

Yep! This has been on my to-do for a long time, actually, but most
bacteriocins are far too large to be carried on a practical cloning
plasmid. Operons run into many kilobases. It just so happens that in
E.coli, there are quite a few very small bacteriocin operons..

In fact, several of them are so small that the primary contribution to
operon length isn't the bacteriocin synthesis cluster, but the
*transporters* to get the bacteriocin outside the cell! Gram negatives
have two membranes, so while Gram+ cells can use generalised export
systems like "sec", E.coli has *no* unified export machinery beyond the
periplasm, and each gene cluster that exports a product has to encode
its own transporter. As it happens, they're all massive!

If everything works *too* well and I have money left over for further
improvement, I've got a speculative method in mind that could
potentially reduce the operon for the bacteriocin to less than ~300bp,
by trying to leverage E.coli's sec system and hack things so the
proteins escape the periplasm anyway.. We'll see how successful the
project is on IndieGoGo before I get ahead of myself! :) But, a 300bp
selection cassette would be a nice hack, I think!

[Koeng]

Out of curiosity, how big is the operon/ transport protein?

Perhaps another hack would be fusing an enzyme to the bacteriocin.... then get it exported

[Patrik D'haeseleer]

In some cases, the immunity protein *is* the transporter. So the ideal case would be to have a small peptide bacteriocin, plus a transporter to provide immunity and secrete the bacteriocin. That may be wishful thinking though ;-)

I'm sure cathal will let us know in good time exactly which bacteriocin he's aiming for - there's quite a few to choose from. I'm assuming some sort of microcin. Those can get as small as a dozen amino acids or less, but most actually require some additional maturation proteins as well.

Meanwhile, there's a bunch of good online resources to explore, including BACTIBASE.

Patrik

[Pieter]

You definitely have my support in developing this! Even though I will have to keep the parts separate in my fridge for a long time to come, I look forward to the day that we are finally allowed to perform a transformation. When that happens, I can't imagine a better experiment than this one.

Everyone involved in DIYBio knows that Cathal is one of the brightest minds in the community. No doubt on my mind that this project has all the right values, motivations and hopefully set a new standard for open biotech.

[Cathal Garvey]

Thank you Pieter! Kind and humbling words, I'm really happy to have you
on board. :)

To anyone who's waiting for a proposed plasmid map, and for more details
on the bacteriocin system, wait no longer:
http://www.indiegogo.com/projects/indiebb-your-first-gmo/x/4252296?c=activity

Happy Saturday!
Cathal

On 25/01/14 09:47, Pieter wrote:
> You definitely have my support in developing this! Even though I will have
> to keep the parts separate in my fridge for a long time to come, I look
> forward to the day that we are finally allowed to perform a transformation.
> When that happens, I can't imagine a better experiment than this one.
>
> Everyone involved in DIYBio knows that Cathal is one of the brightest minds
> in the community. No doubt on my mind that this project has all the right
> values, motivations and hopefully set a new standard for open biotech.
>
> On Thursday, 23 January 2014 04:11:01 UTC+1, Patrik D'haeseleer wrote:
>>
>> In some cases, the immunity protein *is* the transporter. So the ideal
>> case would be to have a small peptide bacteriocin, plus a transporter to
>> provide immunity and secrete the bacteriocin. That may be wishful thinking
>> though ;-)
>>
>> I'm sure cathal will let us know in good time exactly which bacteriocin
>> he's aiming for - there's quite a few to choose from. I'm assuming some

>> sort of microcin <https://en.wikipedia.org/wiki/Microcins>. Those can get

>> as small as a dozen amino acids or less, but most actually require some
>> additional maturation proteins as well.
>>
>> Meanwhile, there's a bunch of good online resources to explore, including

>> BACTIBASE <http://bactibase.pfba-lab-tun.org/main.php>.

--
Please help support my crowdfunding campaign, IndieBB: Currently at
13.4% of funding goal, with 47 days left:
http://igg.me/at/yourfirstgmo/x/4252296
T: @onetruecathal, @IndieBBDNA
P: +3538763663185
W: http://indiebiotech.com

[Jimmy S. Nielsen]

Hi Cathal.
Great idea! I have signed up for the package and hope that you will meet the funding target.
Jimmy

[Nathan McCorkle]

So I started wondering last night, do you have any ideas on how to
cure a strain of such a plasmid? I could imagine someone running out
of wild/plasmidless bacteria, or maybe their lab getting contaminated
with the plasmid and thus causing them to have no more plasmidless
E.coli (plasmidless in the sense of your engineered plasmid).

With antibiotic resistance plasmids, you can usually 'cure' them of
the plasmid by growing them without pressure (no antibiotic added) for
several generations. Is there any way to 'cure' these bugs with your
plasmid? Some 'curative' plasmid that interferes with transcription of
the colicin? Seems like to then get rid of the curative plasmid, it
would have to be controlled externally (i.e. with antibiotics).

Thoughts?

--
-Nathan

[Cathal (Tablet)]

Hey Nathan,
Good question! Curing the plasmid out of the modified strain, to give you the original strain again, would be an interesting project. Because, this isn't just toxin/antitoxin, where if an individual cell survives they can thrive despite the rest carrying the plasmid. Instead, here the plasmid bearing cells will kill any "defectors" fairly quickly!

If you did want to cure a strain, you could try using a temporary knockdown technique to silence one of the proteins that are used by Colicin V to enter the cell. Strains lacking these proteins (not the norm) are immune to Colicin V, so temporarily knocking these proteins down would make a cell resistant to Colicin V long enough to perhaps plate them out, looking for non-fluorescent cells.

Another, harder, way would be to try a continuous flow culture at extremely low culture density. If you replaced the medium at a sufficient rate without washing out the culture altogether, you might keep the concentration of Colicin V low enough for a population of non-bearing cells to emerge. However, consider that Colicin V is considered *extremely* toxic to E.coli cells, with some people reckoning that as few as a single Colicin V protein may be enough to kill a cell without the immunity protein. It forms a pore and just lets the whole cell spill out unless plugged, so even tiny molarities of the toxin would kill all your E.coli on contact..

I wouldn't think this could work so well if it didn't seem such a strong system, so curing it would be, as I said, an interesting project. ;)
Of course, maybe it's trivial, too. Who knows? In wild plasmids Colicin V seems effective enough to make cells maintain it, so I expect the same here.

[John Griessen]

On 01/29/2014 03:33 PM, Cathal (Tablet) wrote:
> Colicin V is considered *extremely* toxic to E.coli cells, with some people reckoning that as few as a single Colicin V protein
> may be enough to kill a cell without the immunity protein. It forms a pore and just lets the whole cell spill out unless plugged,
> so even tiny molarities of the toxin would kill all your E.coli on contact..

They sound like a predatory colony e.g. "the Borg" in a sci-fi scenario. But it's real sci instead!

[Nathan McCorkle]

I guess you'd only need this ability if you were in some trade-control
zone, where getting a strain would be hard/impossible to do. Not sure
if many countries like that exist, but I can think of at least one. Or
maybe in an area where postal delivery from a kind friend is
hard/expensive/unavailable... I can think of more than one country
like this, or remote/undeveloped areas inside countries.

On Wed, Jan 29, 2014 at 1:44 PM, Cathal (Tablet)
<cathal...@cathalgarvey.me> wrote:
> You see, with the right signalling it'd be no problem. But, this is to be a
> 'hackable' plasmid, meaning someone might want to build a project on it that
> requires signals like temperature, tetracycline, etcetera. If I were to use
> those signals as a kill-switch for the plasmid, it'd make the plasmid
> incompatible with projects using those signals.
>
> Synthetic Biology is already short on useful inputs; there aren't that many
> distinct, independent signals you can use at the same time for complex
> projects. Better if I use *none* in the plasmid, and leave them all open for
> people to use.
>
> By and large, plasmid-curing isn't a feature most people want in a cloning
> or engineering plasmid. If you do want it, hack it in; create an inducible
> system with antisense RNA against the toxins and transporters but not the
> immunity protein, and to a lesser extent against the rep protein. So, the
> culture will (simultaneously) stop making toxin, survive for a while, and
> eventually lose the plasmid due to low rep protein levels. But, a killswitch
> like this isn't a feature I'd bother putting into the plasmid on day 1.
>
>
> Andreas Stuermer <masters...@gmail.com> wrote:
>>
>> You would need a silencer RNA that inhibits the toxin gene at 30°C and a
>> replication protein that loses its function at 30°C. That would work well.
>>
>>
>> On Wed, Jan 29, 2014 at 8:23 PM, Andreas Stuermer
>> <masters...@gmail.com> wrote:
>>>
>>> The replication protein can be repressed by tetracyclin? I think the
>>> bacteria can sense tetracyclin long before they actually get strongly
>>> inhibited?
>>>
>>> Or this temperature gene? Above 30°C the replication protein is no longer
>>> produced and thus the plasmid is lost without selection. Actaully - there is
>>> selection. Except if you dilute them very very much. Hm... the toxin will
>>> have a longer shelf-live than the antitoxin - thus if the bacterium loses
>>> the plasmid it gets killed by the remining toxin. That's the sense of it
>>> actually...

>>>> ---------------------------------------------------------------------
>>>> To unsubscribe, e-mail: diybio-eu-...@diybio.eu
>>>> For additional commands, e-mail: diybio-...@diybio.eu

>>>>
>>>
>>
>
> --
> Sent from my Android device with K-9 Mail. Please excuse my brevity.

--
-Nathan

[Nathan McCorkle]

On Wed, Jan 29, 2014 at 2:38 PM, Rüdiger Trojok
<tro...@openbioprojects.net> wrote:
> the interesting part is particularly, that it
> is a wide spread functionality
> in nature, appearing in various forms and concepts. the colicin V system

> It definitively has a bigger survival chance in a wild ecosystem (lets say
> the guts after accidentally swallowing it)
> than a normal AMP marker, which becomes useless without selection pressure.
> Here, the question how common the concept in natural bacterial ecosystems is
> and if it really
> is an advantage compared to wild type bacteria or not.

Very interesting way to look at this.

People are so often worried about spreading antibiotic resistance
genes, and assuming that lack of pressure means the actual lab-strain
E.coli/organism are negligible to the environment (other than by
donating transgenic/advantageous DNA for possible uptake) since they
will soon start to succumb to real-world conditions (as opposed to the
comfort of a lab tech feeding/defending and caring for them).

I've heard of people drinking K-12 E.coli, with no apparent effect,
but would an E.coli with bacteriocin system completely replace the gut
E.coli community?

[Mega [Andreas Stuermer]]

I don't think it could replace your gut flora. You have strains of e coli in there which produce a lot of other toxins and would quikly kill them off. Maybe even such which carry a natural copy of colicinV?

Btw, would it do harm if the e coli in your gut were k12 derivates with GFP? Rather not I gzess. And colicin eon't harm the lactobacilli either?

Regarding antibiotic markers: Amp may be safer. that's the one side of the medal. On the ozher hand, the European Union drasicalliestlly restricts GMO. Self-cloning organism (cisgene) are not regulated. so you either do this or nothing at all^^

[Nathan McCorkle]

On Wed, Jan 29, 2014 at 3:20 PM, Rüdiger Trojok
<tro...@openbioprojects.net> wrote:

> however, the plasmid could be transmitted to other not yet resistant cells
> via horizontal
> gene transfer,

If the bacteriocin system were already present in the existing E.coli
population, why would the bacteriocin plasmid increase the risk of
horizontal gene transfer? Simply because it can diffuse faster and has
greater percentage colicin-to-overall-DNA as a plasmid? Because the
plasmid might be higher copy-number?

[Cathal Garvey]

Well, if they were borg-like you'd expect most wild bacteria to bear at
least one colicin cluster by now, so I don't believe the extreme hype
about toxicity. But, they are pretty damned toxic. :)

--
Please help support my crowdfunding campaign, IndieBB: Currently at

17.2% of funding goal, with 43 days left:

[Cathal Garvey]

Oh good angle! :)

> However, I wonder if it comes to questions of deregulating GM, if
> this system is really a helpful argument - as it is so stable.

> It definitively has a bigger survival chance in a wild ecosystem
> (lets say the guts after accidentally swallowing it) than a normal
> AMP marker, which becomes useless without selection pressure.

Very solid point; without antibiotics, antibiotic resistance is useless,
but colicin clusters are still useful if your main competitor is another
susceptible strain.

The most sensible answer is that it probably isn't a serious concern.
That's because Colicin V is very narrow spectrum; it *only* affects
E.coli, and only if they bear a set of 2/3 targeting surface proteins,
too. It won't help a strain like K12 survive against any other bacteria
that reside in the gut, so K12 won't survive.

So what about other E.coli that might adopt the plasmid? That's more
interesting, because the plasmid might be "loaded" with a payload that
might be not-so-good in the intestines. I'll grant you, although I don't
see it as likely, it's worth considering.

To resolve this, who thinks it *is* worth including a suicide gene on
the plasmid? It could be tet-mediated, so if, by some fluke or
misfortune, you ended up in hospital with a bacterium in your intestines
producing kryptonite, you could suggest a therapy they'll actually have
to-hand in the hospital which will contribute to the success of
eliminating the rogue DNA?

I should stress again that I don't worry about this, and neither should
anyone else. Colicin V is a peptide, and outside of a petri dish
communities of rival bacteria have plenty of ways of destroying
offensive peptides, or evading their effects. It's never even been
studied against biofilm-encased E.coli, which are probably more the norm
in gut flora anyway. This is all speculation, but speculation is fun and
also bears considering for PR and "precautionary" reasons.

On 29/01/14 22:38, Rüdiger Trojok wrote:
> Hey all,
> the bacteriocin functionality is pretty neat. I have suggested a similar
> system in my
> diploma thesis on a bacterial contraception, as it allows to give your
> modified strain
> an evolutionary fitness bonus. the interesting part is particularly, that it

> is a wide spread functionality

> in nature, appearing in various forms and concepts. the colicin V system is,
> as cathal says,
> in the EU considered as selfcloning. However, I wonder if it comes to
> questions
> of deregulating GM, if this system is really a helpful argument - as it is
> so stable.

> It definitively has a bigger survival chance in a wild ecosystem (lets say
> the guts after accidentally swallowing it)
> than a normal AMP marker, which becomes useless without selection pressure.
> Here, the question how common the concept in natural bacterial ecosystems is
> and if it really
> is an advantage compared to wild type bacteria or not.

> Any info on that? Any already made decisions by regulators on it available?
> Best,
> Rüdiger
>
> -----Ursprüngliche Nachricht-----
> Von: Nathan McCorkle [mailto:nmz...@gmail.com]
> Gesendet: Mittwoch, 29. Januar 2014 22:55
> An: diyb...@diybio.eu; pdxhack...@googlegroups.com; diybio;
> diybio-...@googlegroups.com
> Betreff: Re: [diybio-eu] Re: IndieBB Crowdfunding Campaign: Help me make a
> great beginner's kit for DIYbio/synbio!

[Patrik D'haeseleer]

I wouldn't be terribly worried about your plasmid getting out of control in your gut flora. Your gut flora already contains plenty of other plasmids containing colicin V and a bunch of other bacteriocins that are in a constant state of dynamic equilibrium.

That being said, I do think including a suicide gene is a great idea, and could come in handy in all sorts of situations. Showing that the first DIYbio open source novel plasmid design is not only antibiotic free but includes such a safety feature would be excellent PR as well.

Patrik

[Mega [Andreas Stuermer]]

This night I was just thinking about the following: 

What if you put the lux operon into it and somehow get the bacteria in your gut? And, if they would be able to stay in there

Then your intestines would be weakly glowing (but lower oxygen levels would likely limit this) 

The blue light would help stimulate DNA repair, so colon cancer rates may be drastically lowered? 

http://www.ncbi.nlm.nih.gov/pubmed/10708366

http://www.ncbi.nlm.nih.gov/pubmed/17262725

I assume the light induced DNA repair mechanism (via p53) is still present in human cells. 

[Cathal Garvey (Phone)]

Colon is basically anaerobic or microaerobic, you'd get no glow. If anything, the luciferase would eat up what oxygen remained!

[Koeng]

I agree that there should be a toxin switch if it is simple for you to make. 

Also, I had another thought for your campaign. Perhaps make the colicin part biobrick compatible. Try and get iGem teams to fund your project as well, since there is quite a few of them and a self selection method would allow for some awesome biological switches, just what they are looking for.

-Koeng

[Cathal (Tablet)]

Hi K,
I don't know at this point how easy or hard a tet switch will be; I don't have time to do the research as I'm up in Dublin for a few meetings but will catch up when I'm back in Cork.

The crowdfunding campaign is my attempt to get funding from the people who'll use and reshape the plasmid the most. If any iGEM teams happen to have spare cash lying around (?) I'd be very grateful for their support, but frankly I doubt it; I imagine they work to a budget and can't spare the excess.

As far as biobricking the individual plasmid components, that's not really practical while making the multiple-hacking-site biobrick compatible, and it adds overhead that's unnecessary. If someone wants to PCR out the Colicin cluster with biobrick'd primers, that's fine. As per Andreas' great suggestion that I make the individual components of the plasmid easily extractable, I'm hoping to include useful priming sites around each part which would facilitate such an efffort.

I can't let feature-creep bloat this too much, though. I want it to be 'hackable' but that has to be achievable within spec! :)

[Cathal Garvey]

In case ye all feel like chipping in at the last minute, IndieBB is into
the last 55 hours!

In the spirit of last-ditch-efforts, I'm reducing the prices of a bunch
of the "specialist" perks such as consulting and "Your Name in IndieBB".

Please help support my crowdfunding campaign, IndieBB: Currently at

46.3% of funding goal, with 56 hours left: