RE: [DIYbio] Plant chloraplast viruses as vectors

That's a TON of good ideas! Not sure about the inducible autophagy. I
have to admit that since I've been so obsessed with transplastomic
technique, I have embarassingly little knowledge about chloroplast
biology. I know enough to manipulate and hijack some prokaryotic
machinery but little about life cycle.

Im searching for a book by my hero Ralph Bock who underscores all the
important facts about chloroplast and mitochondrial biology of the
plant cell. Its very pricey but may be able to borrow it through
inter-library loan.

The idea of making the chloroplasts secrete a toxin and be resistant to
it if transformed is great but my fear is that it will happen too
quickly and may starve plant of other essential proteins aside from
rubisco that the plant may need and kill the tissue entirely. The
spectinomycin targets 16s ribosome units but the starter dosage during
transplastome selection is relatively tame so it won't kill everything
all at once. Then again the media is supplemented with sugar so
photosynthesis is a luxury. In short, spectonomycin plus secreting
toxins may be too much pressure. Sure as hell want to test your idea
though!

So a construct of such design would look something like this:

HR Flank--Prrn--aaDa--Trps--PpsbA--IndieBB toxin--indieBB
resistance--TpsbA--HR Flank

The flanks would most likely be two transfer rna coding regions and the
construct would be thrown in between as a replacement of the
intergenic "junk" spacer region . Would need to find an area that has
none of the cut sites I use. All minor details.

You could make the indieBB tox antitox circuit polycistronic and throw
in some IEE regions in between the toxin circuit to optimize expression
as an operon and lox sites for good measure. Would need to codon
optimize for chloroplast. Seems doable from a wet lab standpoint. Now
the question is if there is native resistance to indeBB-like toxin.

Maybe invent a protein-based ribosomal repressor specific to
chloroplast? Just throwing thoughts out.


Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D From: Cathal Garvey
Sent: ‎7/‎18/‎2014 8:52 AM
To: diybio@googlegroups.com
Subject: Re: [DIYbio] Plant chloraplast viruses as vectors
I wonder about resolving heteroplastomy.. there's some interesting
tidbits out there about plastid fusions, right? And I imagine autophagy
pathways are as conserved in plants as animals. I wonder if there's
something you could do to your transformant chloroplasts that would lead
them to induce the cell to undergo some chloroplast spring cleaning,
helping to resolve heteroplastomy early?

I know that autophagy can lead to recycling of mitochondria in animals,
I'd expect plastids in plants, too. So, induce autophagy (starvation?
Minor oxidative stress?), and induce mitochondrial damage with something
your transformants are resistant towards (spectinomycin?) so the
untransformed ones are more likely to attract recycling complexes.

Or, study how phages fuse, why, under what conditions, and induce
plastid fusion so there's more cross-over between the plastid pool
within the cell.

Or, pull an IndieBB and make your transformed plastids kill the others
somehow. ;)

On 18/07/14 11:02, Sebastian Cocioba wrote:
> IIRC all of these viral transformations are for transient gene
> expression. I have yet to find a retroviral vector for stable
> transformation in plants. Been pondering about modifying a channel on
> the plasma membrane of a plant cell to act as a phage anchor similar to
> the motifs that bind coliphages. The real trick is the delivery and
> assembly of yet another virus while inside the cell which will bind to
> recombinant chloroplast membranes and actually deliver the target DNA.
> You would still need to have a transplastomic line to modify in the
> first place. Why not just use PEG-mediated naked DNA uptake in
> protoplasts? Tougher to get right but way cheaper than a gene gun and
> will produce stable transformants. Most successful transplastomic plant
> species made using PEG was lettuce.
>
> Also, engineering a virus that infects plants and directly,
> permanently, alters their genome might be hard to contain and could
> pose a scare to the EPA/USDA here in the states as well as elsewhere.
> The thought of an HIV-like particle that can inadvertently infect
> any/all crops may not be the best idea regardless of what the transgene
> is. There are a few patents for universal chloroplast homologous
> integration vectors already filed and there are a number of regions
> homologous to many species/genera along the myriad genes of the
> chloroplast genome so your single virus (if targeting natural surface
> proteins) may infect non-discriminately.
>
> An idea could be to transiently express a channel or membrane protein
> ligand via agro which will be expressed in tandem with a visual marker
> say anthocyanin and then the same tissue is treated with said
> phage-like virus to deliver the DNA goods into the cell. If all goes
> well, a mechanism that is not well understood will cause uptake of DNA
> directly into chloroplast which will recombine, if properly built, to
> form stable transgenic plastids.
>
> After that, one would need to wait and wait for that one transformed
> cell to gather enough chloroplasts resistant to spectinomycin so that
> one single shoot forms. This shoot will be chimeric in nature and have
> a decent amount of wild type chloroplasts still within so the leaf of
> the shoot is chopped up and further cultured until more shoots form.
> This purification process is done several times to ensure homoplastomy.
>
> Last step would be to Cre-Lox out the resistance marker and viral
> ligand sequence via transient agro just to be on the safe side. If all
> goes well you'll have a STABLE virus-mediated transformation of
> chloroplast genome. About a years worth of lab work but a worthwhile
> endeavor.
>
> The current hot topic in plant biotech is plants as biofactories. This
> does not require stable transformants so do sift through the articles
> and ensure that what you are reading is showing evidence of stable, not
> transient, transformations. :)
>
> Sebastian S. Cocioba
> CEO & Founder
> New York Botanics, LLC
> Plant Biotech R&D From: Cathal Garvey
> Sent: ‎7/‎18/‎2014 5:19 AM
> To: diybio@googlegroups.com
> Subject: Re: [DIYbio] Plant chloraplast viruses as vectors
> Can we keep the snarky notes off the refrigerator please? :)
>
> On 18/07/14 07:57, Yuriy Fazylov wrote:
>> Wow. Would you look at that. No violent bursts or air that can take out an eye.
>>
>

--
T: @onetruecathal, @IndieBBDNA
P: +353876363185
W: http://indiebiotech.com

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