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