[DIYbio] Re: AW: [diybio-eu] Re: IndieBB Crowdfunding Campaign: Help me make a great beginner's kit for DIYbio/synbio!

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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:nmz787@gmail.com]
> Gesendet: Mittwoch, 29. Januar 2014 22:55
> An: diybio-eu@diybio.eu; pdxhackerspace@googlegroups.com; diybio;
> diybio-ireland@googlegroups.com
> Betreff: Re: [diybio-eu] Re: IndieBB Crowdfunding Campaign: Help me make a
> great beginner's kit for DIYbio/synbio!
>
> 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)
> <cathalgarvey@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 <masterstorm123@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
>>> <masterstorm123@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...
>>>>
>>>>
>>>> On Wed, Jan 29, 2014 at 8:12 PM, Nathan McCorkle <nmz787@gmail.com>
>>>> wrote:
>>>>>
>>>>> 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?
>>>>>
>>>>> On Wed, Jan 22, 2014 at 12:01 PM, Cathal Garvey
>>>>> <cathalgarvey@cathalgarvey.me> wrote:
>>>>>> 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. :)
>>>>>
>>>>>
>>>>>
>>>>> --
>>>>> -Nathan
>>>>>
>>>>> -------------------------------------------------------------------
>>>>> -- To unsubscribe, e-mail: diybio-eu-unsubscribe@diybio.eu For
>>>>> additional commands, e-mail: diybio-eu-help@diybio.eu
>>>>>
>>>>
>>>
>>
>> --
>> Sent from my Android device with K-9 Mail. Please excuse my brevity.
>
>
>
> --
> -Nathan
>
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Please help support my crowdfunding campaign, IndieBB: Currently at
17.2% of funding goal, with 43 days left:
http://igg.me/at/yourfirstgmo/x/4252296
T: @onetruecathal, @IndieBBDNA
P: +3538763663185
W: http://indiebiotech.com