Re: Plant transformation using Agrobacteirum

[Mega]

Hi Caleb,

 

We have pGreenII plasmid (binary system) here... The JIC (from UK) sells it for I think 40 or 60 british pounds or so.

 

At the moment we are trying to insert the 35S-GFP into it, but it just doesn't wanna work. Blunt end ligation is awful, additionally some of the enzymes in our lab are so old you have to add 30* the usual ammount...

 

That would give pGreenII-0049-Luc-35S-GFP construct...

 

Well, when the Genome Compiler kickstarter campaign runs off, we won't need GFP any more though...

 

Best,

Andreas

 

 

 

Am Freitag, 8. März 2013 01:26:49 UTC+1 schrieb Caleb:

As the title says, this I was I'm looking to do, but one of the main issues I'm having is finding a source that sells the right plasmids for a decent cost. The main gene of interest here is the Green fluorescent protein (GFP, or red, blue, violet fluorescent). I looked on the addgene website, since I had read that that may be a good source, but I wasn't sure if any of the plasmids that they listed had the t DNA function for the Agrobacterium to work. I also looked on the pGreen website, which has plasmids designed for Agrobacterium, but i wasn't able to find a pricepoint on the website, and I wasn't able to contact them at the time. Getting the bacteria shouldn't be a problem, but I am still considering what plant I will use for the experiment. I'm still doing heavy research on the topic, and any feedback would be tremendously appreciated, also let me know if anything needs to be explained better.

[bant...@gmail.com]

Hello Caleb, here at Phytometriclabs we work with many plants that would be ideal for glow in the dark. You would want a dicot with strong white variegation areas as pigmentated areas mask most of the light. If we may be of help don't hesitate to contact us. banta

[Sebastian S Cocioba]

Have you looked into DNA 2.0's CometGFP? They sell open source fluoroproteins as plasmids in bacteria. Decent price point for the "freedom" they provide. Many colors and resistance marker combos. I personally hate pGreen2 and use the pPZP series exclusively for my transformations. As for gfp expression, consider researching a potent promoter like the cotton leaf curl multan virus promoter or the good ol double 35s CaMV promoter. Arabidopsis researchers found a great terminator that works better than the standard Nopaline synthase terminator originally isolated from Agrobacterium. About 17% increase...i guess due to stronger hair pin formation. Anyway try to get your hands on agro line gv3101. Its more aggressive so larger transformation efficency but more easily made resistant due to improper replating frequency aka harder to get rid of. Make sure that its riffampicin and gentamycin resistant. This will ensure the helper plasmid pMK90 is present which has the essential virulence genes for said transformation. Always wear proper gear when working with agro since it can, given the right conditions, attack human cells too. 

I would email labs at universities for a donation of agro but make sure its within your own state. Shipping agro across state lines without a permit is a FELONY. Its considered a plant pathogen and any plants made using agro is regulated due to the fact that its a). Gmo and b). Made using a plant pathogen thus residual bacteria on emerging leaves are still viable (i swabbed transformed plant and regrew agro). Deregulation is expensive so many chose gene guns as an alternative since the law didn't catch up yet thus exemption is easy. This is technically only required if you plan to release the organism. 

If you have access to a pcr machine and a business address to ship primers to, why not build your own agro vector for learning and fun? Its a difficult task due to all the parts u need to source but if you plant to clone using pcr only its much cheaper. Restriction enzymes are expensive. Primers, on the other hand are $5/strand up to 40bp (bless you Value Oligos). All you really need is the agro ori, and the border repeat regions, and kanamycin resostance marker. You could isolate the pmi gene coding for phosphomannose isomerase and make mannose your selection marker (ive done this...it works) and not worry about finding a kanamycin resistance gene. Just amplify the gene from your workhorse ecoli. They all have it. Just keep in mind that while cloning the gene, if you choose to do a diagnostic pcr to see if the gene is there, remember to make your primers so that they bind to the plasmid backbone region just outside the gene and not inside the cds itself so you wont get a false positive when screening ecoli colonies for insert. I learned that the hard way :P

The best advice i can give to anyone wanting to do plant genetic engineering is to start with plant tissue culture first. Depending on the plant it can be a mountain to overcome especially when there is little prior data on somatic embryogenesis of said species. That being said plant tissue culture can be done very cheaply with no kits required. I do all my plant cloning at home using a home made laminar flow hood. Ive also worked with a guy off this list via text msg and helped him get to a functioning cloning lab in relatively short time frame. He had no prior lab experience. Its a good example that anyone can do it especially on a budget. Best of luck and feel free to ask anything regarding plant stuff. I enjoy teaching and sharing info!

Sent from my iPad

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[Cathal (Phone)]

IIRC, last time I looked DNA 2.0 are not *selling* the open fluorophores. They are selling related fluorophores that are not freely licensed.

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

[Cathal (Phone)]

To elaborate, they "opened" some but didn't offer then for sale I think: rather, made sequences public and made a patent nonassertion pledge

[Mike Horwath]

Looks like they are currently selling the four "IP-free" fluorophores as E.coli expression plasmids, although that would not be so useful for plant expression.  The sequences can be found at Biobricks.

[Cathal (Phone)]

Cool, thanks Mike!
They'd need re-synthesis then, IMO, to make them plant compatible. You'd need a codon frequency table for plants and some other parameters, and then you could use PySplicer. /shameless

On 15 October 2014 15:43:49 GMT+01:00, Mike Horwath <mike...@gmail.com> wrote:

Looks like they are currently selling the four "IP-free" fluorophores as E.coli expression plasmids, although that would not be so useful for plant expression.  The sequences can be found at Biobricks.

[scoc...@gmail.com]

What I did was an easy-bake transient expression plasmid to test out my gene gun using CometGFP that I bought from them. I designed some primers to amp out the gfp and added the ncoI cut site which conveniently begets the plant kozak consensus (eukaryotic rbs) and drove the gene with the cauliflower mosaic virus promoter x2. I got decent expression with it without tweaking or codon optimization. If he just needs a source of gfp, and for some reason can't find a lab that's willing to mail him an egfp cassette, this is a viable option. The company will send you a link to their genbank page upon request after purchase.

To be honest, the agrobacterium transformation plasmids contain resistance markers taken directly from bacteria (ecoli transposon for kanamycin nptII) without modification and is expressed and functions as intended in plants. I wouldn't worry too much about codon optimization. With fluorophores, much like peoples attempts at making gfp beer, ph should be taken into consideration. Plants, based on their media requirements alone, prefer a lower ph than ecoli thus folding may be an issue once the protein is expressed. A crude experiment would be to try to grow ecoli expressing gfp on plates ranging in pH and buffered with MES to stabilize around plant media pH and see what happens. Then again, the ph may just hinder ecoli growth and development thus making this experiment crappy. Pardon the mind vomit...

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

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From: Mike Horwath
Sent: ‎10/‎15/‎2014 10:43 AM
To: diy...@googlegroups.com
Cc: scoc...@gmail.com; stygia...@gmail.com; cathal...@cathalgarvey.me
Subject: Re: [DIYbio] Re: Plant transformation using Agrobacteirum

[Yuriy Fazylov]

A lot of people are familiar with GFP but did you check if your GFP has any cryptic introns?

For example clover GFP doesn't.