GUS

itrezzan at pasteur.fr itrezzan at pasteur.fr
Thu Oct 14 11:15:23 EST 1999




On Thu, 07 Oct 1999 13:43:34 +0200, "Gerd " <gerdn at ibg.uit.no> wrote:

>Hello, 
>I am planning to use a GUS (beta-glucuronidase) reportergene i rhizobium,
>nobody around here have any experience - so some tips would help me.

I discovered this forum this week,  and I just read your message.
I try to clone a GUS reportergene also, in fact to insert it into a
recombinant plasmid between a promoter and some lux genes. I have
quite some problems to do it. 
But I heard some things about the assay of beta-glucuronidase:

> 
>I have read that:
> 
>X-gluc is commonly used as the substrate for beta-Glucuronidase in plants
>and on agarplates (blue colour), 

it is more expensive than PNPU, and not quantitative. A method with
PNPU exists for agar plates.

>p-nitrophenyl glucuronide has been used for bacteria in liquide culture
>(yellow 405 nm), 

To use it on plates, you just need to use toluene briefly (remove it
after 5 to 10 seconds), and then to put a drop of a solution of PNPU
(3.2 mg/ml water) on each clone. If the gene of beta-glucuronidase is
expressed, you can see a yellow coloration which appears. Maybe it is
better, for this assay, not to use LB agar plates.


>and 4-methyl umbelliferyl glucuronide to yield fluorescent product.
>
I don't know this substrate.


>So, the questions are: 
>What are the benefits and drawbacks for each of these substrates?
>Does anyone have recipies for concentrations, and assay condidtions for
>these or other possible substrates? 
>I would also love to know how sensitive the systems are to pH, temp., salts,
>whatever....
>

Sorry, I don't know more and would be interested also to improve my
knowledge.

Isabelle

>Gerd 




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