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

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

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.
>>>>
>>>
>>
>