Re: [DIYbio] Re: Chloroplast Transfection?

By default, Agrobacterium is nuclear-targeting, but I think this is due
to targeting factors in the DNA of the transferred plasmid, or just
sheer chance of where the DNA ends up.

One way to get DNA to more reliably target alternative organelles is to
include DNA sequences matching the target sites of DNA binding proteins
found in said organelles, provided the proteins are made in the
cytoplasm. Essentially, you're hoping that as the cell finishes a
protein destined for the Chloroplast, your DNA will be bound by the
protein as the cell moves the protein into the organelle, dragging your
DNA in with it.

So, if your DNA contains valid promoter regions for chloroplasts, you'd
expect to see an uptick in chloroplast transfection already, provided
that some or all of the promoter-binding proteins are made in the
cytoplasm and not inside the chloroplast itself.

If that's not the case, you might have to find some DNA-binding
chloroplast proteins that *are* made in the cytoplasm (bearing in mind
that the genes coding for said proteins might be in the nucleus, not the
chloroplast genome), and shiv in a few sequences that might be bound by
those proteins.

On 18/11/12 07:38, Sebastian S. Cocioba wrote:
> Agro injects into nucleus as far a I know. Normally plastid transformation is dun via gold gun. If you check the plos one article from the guy who made bioglow, it references his choice against agro for plastid transformation.
>
> Sebastian S Cocioba
> CEO & Founder
> New York Botanics, LLC
>
> Sent via Mobile E-Mail
>
> On Nov 17, 2012, at 4:31 PM, Mega <masterstorm123@gmail.com> wrote:
>
>> Hi,
>>
>> as I'll be getting Agrobacterium plasmids quite soon,
>> and there was that paper that said chloroplast transformation using agrobacterium is feasible,
>> do you think it would be worth a try?
>>
>> Basically, I'd use an empty Ti plasmid, PCR a kanamycin resistance and lux operon within it.
>>
>> I think, I wouldn't even need a promoter etc. because the chloroplasts can express operons. So in some of the transformed chloroplasts the gene cassete is inserted correctly between two genes of an operon.
>>
>>
>> Am I completely wrong?
>>
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