Agrobacterium Ti Plasmid ID

[Sebastian Cocioba]

So I managed to isolate and identify a cherry crown gall bacterium as agrobacterium tum. I used the potato disk assay, catalase and oxidase tests, etc and confirmed its agro and it is in fact viable and pathogenic. (agro also has a tell-tale smell)  Now I want to know some info regarding what opine type the Ti plasmid codes for and eventually disarm the plasmid and place my genes of interest into the dna. Basically I want to avoid ATCC charges and have an in-house agro strain for use in plant transformation from wild strains. Does anyone have info or links on the topic of disarming wild strains of unknown plasmid sequence? I'm curious if the left and right nicking borders are the same regardless of strain. Could one potentially use agro's own nicking enzyme to remove the virulent area and ligate some custom sequence along with biobrick cut sites (xba, ecor1, etc) into the plasmid? Side note: wiki says Ti plasmids average 250kbp. Thats huge! Sequencing that would be expensive if the current price is 28-36 cents per bp. Is there a large difference in the various opine coding sites to change the size of the plasmid drastically? Thanks. :)

[Nathan McCorkle]

you might be able to brute force something to a managable sized
plasmid, then get it sequenced and add in an MCS. I'm thinking try a
mini-prep, see if you get decent yields. Shear plasmid and mix with a
resistance/selection cassette (obtained via PCR, then agarose
electrophoresis purification), follow procedure for blunt end
ligation, then electrophorese the ligation reaction and remove all
bands between 1 - 10kb, transform that DNA and look for survivors.
There's a chance it would work, though low... it would be pretty cheap
if you could easily parallelize it. If important elements are on
different parts of the plasmid, you may want to mix sheared DNA
samples (i.e. sheared for short time plus sheared for long time, so
big chunks plus small chunks)

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[Mega]

Actually, I'm ordering a pGreenII and pSoup plasmid. The problem is, I'm doing this together with a lab, and I'm not sure what their opinion is about sharing plasmids. They even pay for it. 

We're in need for a plasmid-less agrobbacterium though (Grow it at 30 or 40 degrees and it will lose its plasmid). Maybe we can make an exchange (depends on the lab personal, as said). 

[Cathal Garvey]

I have my doubts that it will lose its plasmid so easily. Wild plasmids,
with all the necessary bits intact, are quite stable.

Apparently one way of "curing" (the technical term) bacteria of stable
plasmids is to grow them on agar containing an amount of Ethydium
Bromide. Other DNA intercalators may do the same job. You have to grow
them for many generations under these conditions; the precise number
probably vari
es by plasmid etc.

Bear in mind; the technology to use Ti plasmids in Agrobacterium is,
thanks to our horrendously stupid gene patenting systems, patented.
That's why Cambria created alternatives using other species like
Rhizobium, alternatives which are available under a pretty permissive
(but I wouldn't say "open source") license/MTA:
http://www.cambia.org/daisy/cambialabs/3187.html

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[Mega]

Yeah, but the antibiotic resistance of another incompatible plasmid will hammer the other plasmids out? except if it carries the same resistance gene.

[Matt Lawes]

Yes if the origins of replication of the plasmids are incompatible. The problem is you now have the new plasmid in there instead. This approach would work with a temperature sensitive mutation in partitioning genes of the plasmid. Use the TS plasmid to kick out the first one at lower permissive temp with appropriate antibiotic selection, then up shift to the higher temp and grow without antibiotic.

>matt

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[Cathal Garvey]

Ah yes, sorry! That's clever, that could work quite well. Provided you
wait a few generations under selection to be pretty sure of plasmid
replacement, and then cure the "unstable" antibiotic resistance plasmid
by ending selection. Nice!

On 21/09/12 16:30, Mega wrote:
> Yeah, but the antibiotic resistance of another incompatible plasmid will hammer the other plasmids out? except if it carries the same resistance gene.
>

[Matt Lawes]

I got skills, lol. Card carrying PhD microbiologist / bacterial geneticist....... more of a gentleman scientist these days so DIYBio is a cool ethos to me!

>matt

Sent from my Verizon Wireless 4G LTE DROID

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From: Cathal Garvey <cathal...@gmail.com>
To: "diy...@googlegroups.com" <diy...@googlegroups.com>
Sent: Fri, Sep 21, 2012 12:22:53 EDT
Subject: Re: [DIYbio] Re: Agrobacterium Ti Plasmid ID

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[Nathan McCorkle]

On Fri, Sep 21, 2012 at 12:39 PM, Matt Lawes <ma...@insysx.com> wrote:
> I got skills, lol. Card carrying PhD microbiologist / bacterial
> geneticist....... more of a gentleman scientist these days so DIYBio is a
> cool ethos to me!
>

*Like*

[Mega]

Hi guys,

I was just wondering  how much DNA you can put into a pGreen plasmid before it gets instable...

Can you, if the pGreen has roughly 4kbp, add around 10 kbp between the Ti-borders without fearing that it gets unstable, does recombination, etc. ?

We would then have ~ 15kbp in total, (say worst case up to 20kbp). Will the problem be transformation rather than stability?

 
Best,

[Nathan McCorkle]

I think the problem comes with transformation efficiency, using phage recombination proteins you can just transform linear DNA with resistance+custom DNA to knock out a section already in the e.coli.... that section could be another plasmid that you already transformed (using a different resistance marker)... then you can eventually wind up with a bacterial artificial chromosome if you keep adding sections like that

http://en.wikipedia.org/wiki/Recombineering

http://en.wikipedia.org/wiki/Bacterial_artificial_chromosome

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[Andreas Sturm]

Ôk, but if I get the plasmid inside, it will be quite stable in sense that it is not recombinated or something like that?

With 15 kbp, it should still be possible, althought efficiency may be bad (?)

[xmort]

From my experience with pGreen the size of the insert shouldn't be a problem. We use these plasmids with inserted  full-length infectious cDNA copy of  plant viruses (PVX or TMV). The PVX is the longer one  however  its more stable than the shorter TMV.  PVX insertion with promoters and other regulatory sequences is around 10 kb, TMV about 8 kb. I think that the stability or instability is dependent on the inserted sequence features. 

There are several things you might do when you think that it became unstable. First lower the temperature and kanamycin concentration. We use 25 µg/ml instead of 50, we incubate plates and tubes at 28°C instead of 37~C. Top10 cells seem to be better than DH5a or MC1061. Didnt try JM109 though. 

It is much more stable  in Agrobacterium than in E.coli, most likely because of different copy  number.  So if you have good competent Agro, you might try to clone it directly to Agro, but it might then be difficult to verify the correct clones. 

There are few issues related specifically to pGreen, or  at least to some versions of it.  There is an "undocumented"  insert of  about 800 bp between SaRep and Kan selection marker. This is E-coli genomic DNA, so it might increase the tendency to recombine with genomic DNA in E.coli.  Also Kan marker might be in opposite orientation than some of the maps show. This might be important, since I traced down the instability to inefficient transcription termination of Kan gene in E.coli and transcriptional read-through to the   T-DNA insert. In other words the same inserted DNA can be unstable in one orientation and pretty stable in the other. But your mileage can vary:)  

So it might be a good idea to play a little with your version of pGREEN to confirm that your map and the plasmid are identical. 

[Andreas Sturm]

Sounds great, thanks!

Oh, 800 bp is quite a big potential for recombination...

On Tue, Dec 18, 2012 at 10:19 AM, xmort <mor...@ueb.cas.cz> wrote:

.  There is an "undocumented"  insert of  about 800 bp between SaRep and Kan selection marker. This is E-coli genomic DNA, so it might increase the tendency to recombine with genomic DNA in E.coli.  Also Kan marker might be in opposite orientation than some of the maps show. This might be important,

But I don't think we can directly transform it into agrobacterium with the PEG transformation methode because efficiency will be quite low...? On the other hand, if we "just" get three transformed colonies, we have three colonies, of which one may have the insert.
Or we get the electroporator which is in one of my university's labs, but this was kind of plan B.

However, we could try it. The betaglucosidase on X-Gal will show where the insert is, it may work in agrobacterium too (gram negative)...

[xmort]

Electroporator will be necessary for direct transformation of ligation mixture to Agro. If it is available at the university you should be OK. However I would experiment with direct agro transformation only if it really shows great instability in E.coli. It might be and should be OK though.

Dne úterý, 18. prosince 2012 16:39:56 UTC+1 Mega napsal(a):

[Andreas Sturm]

Thanks for the info!!

Well, I saw that there were *loads* of electroporation cuvettes in a proffessor's lab, so we 99% have one.

But, for easy repeating, we'd like to try PEG transformation first... It works with all kind of cells... In case it doesn't work with PEG, we try it with e.poration the.

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