Wild-type organisms suitable for plasmid extraction?

[jarlemag]

Hello,

As part of setting up a DIY bio lab and introducing newcomers to molecular biology, it would be nice to demonstrate plasmid extraction and subsequenct study of the plasmid by restriction digestion or sequencing. The catch is, local regulations prevent us from working with genetically modified organisms, including organisms transformed with engineered plasmids. Are there any wild-type BSL1/generally recognized as safe organisms harboring natural plasmids which might be suitable? A strain that is readily available from a culture collection or from fellow DIY biologists would be preferable.

Best regards,

Jarle Pahr 

[Cathal (Phone)]

Natto is fermented with B.subtilis natto, many or most strains of which carry one or two plasmids. The plasmids I'm familiar with are low copy number though so aim for high cell titers. Also, gram positive near-wildtypes means you'll need lysozyme!

I've never done it, but it might work. And not just BSL1 but foodgrade/GRAS. :)

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[Koeng]

Apparently E coli nissle has 2 plasmids

http://www.microbecolhealthdis.net/index.php/mehd/article/viewFile/7512/8855

In Europe you can get E coli Nissle 1917 from the probiotic "Mutaflor". You could use basic plasmid miniprepping reagents that companies are always trying to sell. I don't know about copy number, though.

-Koeng

[jarlemag]

Thanks for the tip, that's interesting. From what I'm reading after some googling it seems that Mutaflor is not available for sale in Norway though, and might not be legal to import privately due to being considered a pharmaceutical. Maybe it's possible to get the pure strain somehow.

Best regards,

JP

[Keoni Gandall]

I can send you a sample if you want.

-Koeng

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[jarlemag]

Thanks for the offer - maybe later? We don't have a lab space for microbiology/culturing set up yet.

Best regards,

JP

[Mega [Andreas Stuermer]]

Do you need a lab for this? Some countries just won't care as long as it's not GMO. You can use a pressure cooker, incubate it on a bookshelf and buy a miniprep kit and do gelelectrophoresis in the garage. Avoid Ethidium bromide though.

[jarlemag]

Not legally speaking no, probably. Personally I want to take the 'high and dry' route though, to stave off concerns and possible negative attention. 

Also, part of the point is to do it in a 'public' setting (we are based in a local makerspace at the moment), so someone's home isn't suitable for that.

Thanks for chipping in. I never have worked with Ethidium bromide/UV transillumination, and never will if I can help it.

[Mega [Andreas Stuermer]]

Ethidium bromide isn't crazily toxic I believe, I have heard of stories of people touching it with bare hands accidentally and still having their hands. Also it is used intravenally in cattle against parasites I heard. But cancer may come as long-term result.

But, there are way safer alternatives like Midori Green which also intercalate with DNA but pass cell membrans hardly. So better go with that.

[Koeng]

http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1001226

"many would be shocked to learn that EB is still used for treating cattle that provide beef and milk for human populations"

It's not that bad, but it is pretty cheap. GelGreen is a good completely safe alternative (cannot go through membrane), however, there are some reports that it changes small DNA mobility in a dose dependent manner because of its binding (which sort of makes sense since it cannot penetrate through the plasma membrane). That doesn't really matter to me because I just need verification gels of PCRs of large fragments, but it might not be the best choice if you need to see the size of small fragments. 

-Koeng

[Francis Lee]

Derek Lowe wrote a nice post about this earlier this year:

http://blogs.sciencemag.org/pipeline/archives/2016/04/18/the-myth-of-ethidium-bromide

On Wed, Jul 27, 2016 at 5:22 AM, Mega [Andreas Stuermer] <masters...@gmail.com> wrote:

Ethidium bromide isn't crazily toxic I believe, I have heard of stories of people touching it with bare hands accidentally and still having their hands. Also it is used intravenally in cattle against parasites I heard. But cancer may come as long-term result.

But, there are way safer alternatives like Midori Green which also intercalate with DNA but pass cell membrans hardly. So better go with that.

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