Anybody has E.coli lactose operon in a plasmid?

[Dorif]

Hello!

I want to make more potent rodenticide from Salmonella enterica ssp. enteriditis var. Issatchenko (non-pathogenic for humans and other mammals - rodents only. It is used in rodenticide called "Зообакцид", from which it was isolated) by "learning" it to use lactose as carbon source (Salmonella sp. can not use lactose). I presuppose, what E. coli is quite similar with Salmonella sp. in genetic plan and these genes will work in it. So, I need lactose operon from E. coli or at least LacZ and LacY genes from it. I have BSL 2 lab at Moldova State University and rats from vivarium for this work, so it will be secure.

Can anybody send me a plasmid with needed genes?

Thanks for help!

[Mega [Andreas Stuermer]]

Hi! Maybe the easiest way is to get it from PCR of (cooked at 90°C for 5 mins) diluted stool sample?

Bare in mind that you might need am ethic votum to work with vertebrates

[Dorif]

In my country it is not the easiest way, because at us PCR is not well developed.(

And, in my country people work with vertebrates without ethic votum - rats are freely used for dissections at the Faculty of Biology and Medical University. Different standards in different countries.

[Nathan McCorkle]

On Mon, May 25, 2015 at 10:56 PM, Dorif <dor...@gmail.com> wrote:
> In my country it is not the easiest way, because at us PCR is not well
> developed.(

In that case, what will you do with the operon once you get it? Try to
induce horizontal gene transfer? I wouldn't try that with potentially
pathogenic samples... get some kind of stripped-down plasmid with
otherwise non-scary genes on it... you don't want dead/half-dead rats
to become a carrier and spreading genes that aren't helpful for
humans.

[Simon Quellen Field]

On Tue, May 26, 2015 at 9:13 AM, Nathan McCorkle <nmz...@gmail.com> wrote:

you don't want dead/half-dead rats

​Shrodinger's Rat?​

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[Matt Lawes]

From a technical angle, the lactose operon (lacZYA) is rather large. Cloning plasmids with blue /white screens use a fragment (alpha)  of  lacZ and the rest on a propagate (lambda). So I doubt you'll find a plasmid source.
>matt

Simon Quellen Field <sfi...@scitoys.com> wrote:

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[Matt Lawes]

Propagate should read prophage. Silly autocorrect, lol.
>matt

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[Nathan McCorkle]

On Tue, May 26, 2015 at 9:19 AM, Simon Quellen Field <sfi...@scitoys.com> wrote:
>
> On Tue, May 26, 2015 at 9:13 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
>>
>> you don't want dead/half-dead rats
>
>
> Shrodinger's Rat?

Heh heh, I was thinking more along the lines of the rodenticide not
working fully, and the rats walking around and transferring some
(possibly) impure rodenticide extract littered with pathogenic (to
humans) genes for horizontal gene transfer. Probably would only create
a problem in a sci-fi movie scenario, but it seems like an easy
up-front workaround.

[Nathan McCorkle]

On Tue, May 26, 2015 at 9:24 AM, Matt Lawes <ma...@insysx.com> wrote:
> From a technical angle, the lactose operon (lacZYA) is rather large. Cloning
> plasmids with blue /white screens use a fragment (alpha) of lacZ and the
> rest on a propagate (lambda).

(reading it with prophage)

I thought the rest was intact in the E.coli genome, with just lacZ
being knocked-out.

[Dorif]

I'll place it in a plasmid and transform Salmonella with it. If plasmid will be too large - I'll use electroporation. And, as I mentioned before - it is not pathogenic to humans.

[Matt Lawes]

Yes in regular e coli lac Opteron is there intact. In cloning strains there is typically a deletion of the region containing lac operon and a lac Z (omega, I.e. alpha deleted) YA cassette added back in lambda prophage.
>matt

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[Matt Lawes]

Two issues.
#1 large inserts in plasmids are typically genetically unstable (due to copy number / potential for rearrangement or deletion). Coli and salmonella genomes will not recombine unless you use special "mutator" salmonella strain. 
In any case you are quickly stepping into bacterial genetics PhD program level manipulations. (My background). Chances for success are low, but not impossible. Risk of much time wasted though is very high.
>matt

Dorif <dor...@gmail.com> wrote:

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[Dorif]

Well, virtually I don't need entire operon for this. It is enought to insert LacZ and LacY. If LacZ is functional with deletion in it - it is reaaly good idea to use this "cutted down" LacZ to minimize the insert size.

[Nathan McCorkle]

On Tue, May 26, 2015 at 11:09 AM, Dorif <dor...@gmail.com> wrote:
> Well, virtually I don't need entire operon for this. It is enought to insert
> LacZ and LacY. If LacZ is functional with deletion in it - it is reaaly good
> idea to use this "cutted down" LacZ to minimize the insert size.

I think Matt's point was that in common plasmids, the lacZ is not
complete... this is for alpha-complementation, since the lacZ gene
product (the protein) is a tetramer composed of two dimers, one of
which is alpha and the other omega... so these common plasmids usually
lack one half of the lacZ gene (which I'm guessing is actually
polycistronic, e.g. two genes in one).

See here for a nice paragraph and graphic of the protein subunits:
http://www.ncbi.nlm.nih.gov/pubmed/23011886

[Dorif]

What about this plasmid: https://www.addgene.org/13422/ ? It seems, what it has complete LacZ, doesn't it?

[Nathan McCorkle]

Looks complete from a length perspective (gene is 1024 AAs, 3 nucleotides per residue, 3607 - 539==1022.66 which is close enough to 1024... Assuming 1024 includes both regions).

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[Dorif]

So, it can be combined with LacY from IGEM in a plasmid and transduced in Salmonella. But how to make the plasmid more stable with this huge insert? I know, what exist some stable big plasmids, what encode pathogenity factors or pathogenity "enhancers" at some bacteria (pPCP1, pMT1 in Y. pestis, pXO1, pXO2 in B. anthracis), but what makes them stable?

[Mega [Andreas Stuermer]]

Bacterial artificial chromosomes or toxin-antitoxin systems?

[Александр Дориф]

It is currently used as rodenticide. No one will use human pathogen as rodenticide.

2015-07-11 0:46 GMT+03:00 'SC' via DIYbio <diy...@googlegroups.com>:

Why do you think this strain is not pathogenic for humans?

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[Cathal (Phone)]

"No one would use a substance toxic to humans as a rodenticide"

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[SC]

""No one would use a substance toxic to humans as a rodenticide""

Lots of rodenticides are toxic to humans.  I wouldn't put rat poison in my tea.

Do you have actual reason to believe this strain is not toxic to humans?  

[Александр Дориф]

It is used without further thermal treating of protected products. So, it is harmless for us. And there is major difference between chemical and biological poisons.

And even if it is toxic - it doesn't matters, because we have good biosafety equipment and can safely handle much more interesting things than Salmonella.

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