[DIYbio] Re: Plant chloraplast viruses as vectors

One of my ideas was to import a natural competence system into the mitos or chloros but that'd be pretty difficult and take quite a bit of genetic engineering to the point where its probably not worth it. But hey, it'd be pretty cool to be able to transform any DNA you want and have it recombine into the plastids (if it has homology)

Btw, does anyone know any self selection mechanisms that could be used in mitochondria or chloroplasts? A selfish gene element that spreads itself perhaps, like I-SceI meganuclease. (Its an intron in yeast mitochondria that cuts the tRNA gene. Only when I-SceI's gene is inserted will it stop cutting). That way it could spread throughout the mitochondria, because as you guys know there are like 20-50 mito genomes per cell. (I've only looked at yeast). Or, if you are going for a plasmid system, that's also very possible. The problem with mitochondria is they use really really weird machinery, both transcriptionally and translationally. 

http://www.pnas.org/content/77/6/3167.full.pdf

Yeast mitos use a ton of wobble codons, which makes the tRNA compact, but also changes some of the codon usage rules, so every gene has to be synthesized and recoded.

http://nar.oxfordjournals.org/content/26/3/689.full

Apparently, there are some retrotransposons in yeast mitochondrias, which could be used for a selfish gene

http://www.sciencedirect.com/science/article/pii/0300908496847560

Probably a more amazing thing would be RNA import into the mitochondria. Don't quote me on this, but I heard of a lab that tried doing this with Cas9. They used a Cas9 with a mitochondrial localization tag to try and engineer the genome and I think they said the sgRNA fell off. I don't know though. http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/targetron.html these things encode an mRNA, but on that mRNA is a protein and this intron element. The protein binds to the intron element and looks for DNA to target for insertion of the element. They've found it to be "retargetable" so you can target new genes. (Mitochondrial localization tag + retarget for the chloroplasts/mitochondria. Recombines in, then make it act as a selfish gene element to transform itself into the rest of the genome copies). Anyway, going back to the RNA transport, it may be possible to import the sgRNA for CRISPR with that mechanism, and then put Cas9 on a mitochondrial localization tag, and then you get mitochondria engineering!

(another article on it) http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035321

(and another) http://schneider.dcb.unibe.ch/PDF/publications/Schneid-TCB02.pdf

I might try the "targettron" sometime if I had more money. They literally charge a thousand bucks for the plasmids I want http://www.sigmaaldrich.com/catalog/product/sigma/tv0010?lang=en&region=US , after tax and shipping. http://www.sigmaaldrich.com/catalog/product/sigma/t2826?lang=en&region=US This one is a bit cheaper. I have designs for it in yeast, but haven't actually gone to construct the plasmid. But hey, if there's anyone out there that wants to try my idea on chloroplasts I'd be more than happy to help. 

Going back to the natural competence idea, here's a good review on natural competence with single membrane bacteria (Bacillus subtilis) http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2009.00164.x/full . I gotta go look for another reference, but there's a similar mechanism in Acinetobacter baylyi, a gram negative. (double membrane!) Acinetobacter baylyi is an easy-to-use bacteria that is biolevel safety 1. If you could combine the genes together somehow (protein fusion or polycistronic expression, with the export machinery to the next membrane) and gene gun that big plasmid into chloroplasts or mitos, just maybe you could get the natural competence to be expressed. If it's expressed, genetic modification becomes easier than modifying the genome. The recombination is really cool because you could actually have plasmids in the cytoplasma or nucleus that uses it's awesome repair system, get imported into the mitochondria, a constant mechanism of repairing mitochondria. (Possibly help aging?)

Anyway, those are just some of my ideas. I think the targetrons could work better than phage. With phage, you'd have to go through genetically engineering both the phage and the mitochondria or chloroplasts to get it in there. With the intron/targetron method, not only do you get constant pressure for recombination, you have an enzyme that will site specifically recombine things for you. And even better, if expressed in the genome there will be constant pressure for the plastids to uptake your DNA (the system goes through an RNA intermediate though, mRNA -> Protein, mRNA  -> Protein finds site -> protein does reverse transcription-> DNA inserted), therefore no selection at all is required. Even better, you can recombine anything you want into it, as the intron has an actual location you can clone things into for site specific recombination! But there is a large "scar" region in that method, but you can recombine lox sites or CRISPR sgRNAs to get whatever you need for downstream engineering in.

Things I think could definitely go wrong:

RNA no bound to protein strong enough, gets knocked off when going into mitochondria (heck like a 100bp of RNA got knocked off Cas9)

RNA intron can't recombine that big of DNA, therefore gets stopped

Just my crazy idea. I might try cloning my construct when I get time. If anyone is willing to try my constructs in plants, contact me. Sorry for rambling :)

-Koeng
(Hopefully someone doesn't take my idea xD)
On Thursday, July 17, 2014 10:47:56 PM UTC-7, Yuriy Fazylov wrote:

Can a plant virus or a phage virus with recombinant chloraplast ligands be used to shuttle in genes?