Re: [DIYbio] Re: IndieBB Campaign Survey; Help Appreciated

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I'd be a bit more cagey on the original question. Yes, if you knew what
genes you wanted, you could use IndieBB as an intermediate vector to
contain the DNA while you wrangle it into the right shape.

This is the normal way of engineering plants; first assemble and prepare
the DNA in bacteria, usually E.coli on a special Agrobacterium shuttle
plasmid, then transfer to Agrobacterium, then get Agrobacterium to
infect plant tissue with the plasmid, and then try to regenerate an
entire plant from modified tissue.

You *could* do this with IndieBB, but if plants are the goal perhaps
you'd be faster pestering Sebastian. That's because in addition to your
desired DNA, you'd need to add the DNA for plasmid housekeeping
functions in Agrobacteruim, and the special bits that Agrobacterium uses
to seize your plasmid and shove it through a molecular needle into the
plant tissue.

As far as "maximum size", yes; most plasmids have a defined carrying
capacity, beyond which they become unstable. I'll be aiming for larger
carrying capacity over copy number when choosing the origin for IndieBB,
because the trend with synbio is towards more complicated genetic
systems..but I won't be going for the real heavy-carrying ones, because
they're often also low-copy-number and therefore possibly harder to work
with using DIY methods.

I don't have time to dig deeply into the research (though there's a
pledge for that!), but I'd imagine that the gene system in Spathiphyllum
and other "air purifying" plants is opportunistic and not active,
perhaps not even specific to the pollutants it's associated with. That
is, the plant's stoma are constantly allowing air to diffuse in and
around the leaf tissue, and volotile organic compounds diffuse into the
cell cytoplasm; there, it's just converted by a cytochrome or other
enzyme into something the cell can use, or is just detoxified and dumped.

If this is the case, and if someone has found the appropriate protein,
then you could probably get a bacterium to express that cytochrome
(watch out for cofactors bacteria mightn't have much of) or enzyme, and
then grow them in liquid broth with air sparged in through as fine a
stone as possible. VOCs will dissolve in the broth as readily as they do
in plant tissue, and get converted, digested or fixed by the bacteria.

Downside; the output will smell of bacteria, so you might want to use
indole-negative E.coli and add the gene for wintergreen or "fresh rain"
to make your air purifier also produce perfume. Making pseudo-defined
broth medium that doesn't smell funny would be a little bit of work, but
not too much I imagine. :)

On 22/02/14 19:17, Dirk Broenink wrote:
> Cool, thanks. I guess I would need to know which part of Spathiphyllum's
> DNA codes for 'air cleaning' and which doesn't. Is there a maxium size of
> base pairs that can be put into plasmids?
> Another question, where do I buy restriction enzymes and ligase, and how
> much would that cost? Can I ship that to The Netherlands without problems?
>
> On Saturday, 22 February 2014 18:38:29 UTC+1, Mega [Andreas Stuermer] wrote:
>>
>> Hi Dirk,
>> In theory yes. The kit allows you to take the "air cleaning genes" and put
>> it into the plasmid (but you need to buy the restriction enzymes and ligase
>> yourself). if you incubate plant protoplasts with PEG and DNA it will take
>> up the DNA.
>>
>> You could do that with any other plasmid too. But this gives you the
>> advantage of avoiding antibiotic resistances.
>>
>> On the other hand, the immediate target of the kit is to learn the basics
>> of DNA work in bacteria.
>>
>>
>

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