PEG transformation - plasmid size

[Mega]

Hi all,

Has anyone experience, how big a plasmid can get, so that you still can insert it into E.Coli / Agrobacterium using PEG transformation? Or is this no problem anyway when supercoiled?
When using electroporation, can the plasmid be bigger? 

Are up to 16 kbp ok?

Is there recombination problems with E.Coli? (If no genomic DNA of E Coli is in the plasmid)

[Sebastian S. Cocioba]

Think of the actual diameter as a function of increasing circumference. I used 20k+ plasmids routinely. There is not much change in its size. Supercoiled is just long but only a few angstroms wide. No biggie. Let me know if you need peg help. We can tandem experiment if you need concurrent measurements/observations. 

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

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[Sebastian S. Cocioba]

Why not just heatshock the plasmid into the agro. 30 sec at. 42 works great for my 18-20k+ Ti plasmids.

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

On Feb 3, 2013, at 4:24 AM, Mega <masters...@gmail.com> wrote:

[Andreas Sturm]

Thanks for your help!! 

And about recombination deletion or mutation, is this an issue? I mean if there is no genomic E coli DNA. Copy number is high. 

You mean the heat shock with liquid nitrogen for agrobacterium? Or the usual E.Coli procedure for agrobacterium - never heared that would work... ? 

[Cathal Garvey]

Recombination in E.coli depends on the strain, the plasmid, and the
plasmid content. If you're using recA- strains, which are lacking a
recombinase gene, then DNA is generally pretty stable. If you're using a
plasmid that isn't usually stable with large inserts, then you can
expect trouble. And, the larger the insert, the more opportunity there
is for the plasmid to find suitable sites for recombination that lead to
deletions; that's just plain statistics, but it mightn't be significant
enough to worry about.

You can reduce your odds of gene loss by designing your gene to include
plasmid or gene maintenance stuff; you've been looking at
toxin/antitoxin systems in another thread, you could make those part of
your gene cassette, possibly even part of the operon you're designing.
If you're synthesising a plasmid from scratch, you could even include
your resistance marker (if any) in the operon, too.

Regarding Agrobacterium transformation, Sebastian would know more. I
think you can do E.coli-style heatshock on Agrobacterium, I haven't
heard of transformation using Nitrogen before..

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[Andreas Sturm]

Ok, thanks. 

I looked up heat shock transformation of Agrobacterium, and just found the procedure using liquid nitrogen. 

So I assumed you can't do a normal heatshock. Therefore I plan using PEG transformation. 

For the  toxin/antitoxin systems, do you know by chance a suitable promoter for E.Coli, that has good expression levels, but not super-high viral expression levels? 

[Sebastian S. Cocioba]

What? LN2? Thats awesome but is there any specific reasoning for that? I've always just used a hot water bath and its just a bit of experimentation for the perfect exposure time since tube density, cell density, and uniformity of water in the bath may vary. Just streak on selection and compare colonies. My go to temp is 42 for 30sec. Average plasmid size is aboot 18k. Just try not to go over 42 unless you feel like trying it. It may be too much for the bug to handle. PEG for transforming bacteria seems overkill but by all means if you can get more efficiency then go for it. Do let us know if your peg method beats heatshocking.

As for peg, I'm not sure it can bridge peptidoglycan. Normally peg is an agent of aggregation so it can bring the plasmid and the bacteria closer but I cant see it working on its own. Do you have a ref for this? Normally peg is used as an alternative to agro and not as a means of transforming agro itself. 

PS for your library I would recommend plant transformation technologies, the book. I think it would help you guys a lot since it covers particle and agro very well and is sourced from the labs that pioneered the tech. Its a bit pricey since its a Wiley Press product but well worth it.

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

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[Andreas Sturm]

http://www.pnas.org/content/86/7/2172.full.pdf

PEG transformation of E Coli. 

http://www.researchgate.net/post/What_is_freeze-thaw_method_for_transformation_of_agrobacterium

LN2 freeze-thaw method agrobacterium.

If it works with the water bath also that's great. You'll need CaCl2 for it? And grow agrobacterium into exponential phase before transforming? 

[Andreas Sturm]

Sebastian, do you usually screen Agrobacterium after transforamtion? Or do you just rely on that they haven't changed the insert after taking it up? 

I just came across an article that said it is essential. However Tomas meant, > 9 out of 10 should be ok. But he recommended screening anyway. 

What's your opinion on that?

[Sebastian S. Cocioba]

Screen in what sense? PCR and gel? Sry I'm confused about the screening since up to this point I thought insert manipulation was low to the point of negligible. I would love to read that article and instill some new found paranoia into my routine :P. i love it when more variables get introduced and the game gets harder. Yay, better science!

Sebastian S Cocioba

CEO & Founder

New York Botanics, LLC

Sent via Mobile E-Mail 

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[Andreas Sturm]

Yeah, in sense make a miniprep of the agrobacteria and cut the plasmids, run it on a gel to confirm that the agfrobacteria haven't mutated / lost part of the insert.

It appeared when looking for agrobacterium transformation on Google Books - in one of the preview pages this was written...
Gonna try to find it again.