Does anyone have pKLAC2 to share?

[Mega]

Hi, 

I'm keen on marker free selection and was just wondering if anyone had this awesome plasmid to share... 

Could give something in return, such as pGreenII. p35S-GFP, pVIB from a brand new midiprep that I got a lot of glowing colonies with, ... On filte paper or in an eppi. Or some other compensation ;) 

[Nathan McCorkle]

I'd planned on using this too, I had some, but left it at school in
their -70C freezer some 3000 miles away from me now :(

It might still be there, but I don't plan on going back anytime soon.
I might be able to have a friend check.

I never used it because I didn't have the time to do all the cloning,
but I'd love to see it spread around the DIY community so I can get my
hands on it when I've got the time to play.

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--
-Nathan

[Andreas Sturm]

Yeah, a pity... It costs more than 300€ when buying it officially...

With this plasmid DIY bio (in yeast) would be much more sustainable than traditional engineereing with antibiotic markers :D

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[Avery louie]

What is pklac2?  What kind of screening does it use?  blue white screening?

--A

[Avery louie]

or is it for use in knockout yeast?

--A

[Nathan McCorkle]

You can use it in k.lactis for sure, but it probably will work in s.cereviseae too (maybe the lac4 won't undergo homologous recombination)... I think it has the '2 micron' ori and selection might include antibiotic (for use in e.coli before final transformation) but definitely acetamidase

https://www.neb.com/products/n3742-pklac2-vector

[Andreas Sturm]

https://www.neb.com/~/media/Catalog/All-Products/6F454BB8401043D798342A0719273945/Datacards%20or%20Manuals/N3742Datasheet-Lot0011210.pdf

It is an integrative yeast plasmid. It secretes the protein of interest into the medium. 

And, best of all, it makes an enzyme that gets nitrogen from an artificial nitrogen source. If you plate the yeast on nitrogen-deficient medium, only those can grow... 

[Andreas Sturm]

AFAIK, it doesn't have a yeast origin, so it must integrate into the yeas genome for propagation. 

[Andreas Sturm]

And the ampicillin resistance for selection in Coli doesn't integrate into the yeast genome. 

[Andreas Sturm]

Still one thing: 

Perhaps (likely) it will work in Cervisiae too. 

But if you want to produce medicine (such as EPO :D ) you will need a yeast that has a glycosyllation that resembles the human glycolysation. 

And K.Lactis is much closer to tthe human than S.Cervisiae. 

[Nathan McCorkle]

On Mon, Mar 18, 2013 at 5:25 AM, Andreas Sturm <masters...@gmail.com> wrote:
> AFAIK, it doesn't have a yeast origin, so it must integrate into the yeas
> genome for propagation.

Seems you're right:
https://groups.google.com/d/msg/diybio/1GHO29KF1VY/Jl9zz3VztXIJ

--
-Nathan

[Conner Berthold]

I should be able to get it for you , but it may be a week or two. Send me an email at... cbertholdbio at gmail dot com

-Conner

[poli]

If you are just interested in the acetamidase gene you could consider having it produced synthetically by idt. They will make qblocks - custom 500 bp oligos for $99 if its longer just make it in two sections and ligate, fusion PCR, or gibson them together. I am planning to do this with a codon optimized acetamidase to use in my organism.

On Sunday, March 17, 2013 4:03:08 PM UTC-4, Mega wrote: