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

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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>.
>>
>> Patrik
>>
>> On Wednesday, January 22, 2014 6:44:05 PM UTC-8, Koeng wrote:
>>>
>>> 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
>>>
>>> On Wednesday, January 22, 2014 5:33:23 PM UTC-8, Cathal Garvey wrote:
>>>>
>>>> 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!
>>>>
>>>> On 23/01/14 01:27, Patrik D'haeseleer wrote:
>>>>> 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
>>>>>
>>>>> On Wednesday, January 22, 2014 3:51:46 PM UTC-8, Cathal Garvey wrote:
>>>>>>
>>>>>> 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 <koen...@gmail.com <javascript:>> wrote:
>>>>>>>>>
>>>>>>>>> "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
>>>>>>>>>
>>>>>>>>> On Wednesday, January 22, 2014 12:01:00 PM UTC-8, Cathal Garvey
>>>> 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. :)
>>>>>>>>>>
>>>>>>>>>
>>>>>>>> --
>>>>>>>> Sent from my Android device with K-9 Mail. Please excuse my
>>>> brevity.
>>>>>>>>
>>>>>>>
>>>>>>
>>>>>
>>>>
>>>
>

--
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