IPTG concentration for induction?

[Daničle EUSEBE]

I have to induce beta-galactosidase in a liquid culture of E.coli Y1089
lysogen for a lambda gt11 phage containing a beta-galactosidase-fusion
protein gene.
I have read in a protocole to use 10 mM IPTG, which seems to me an
enormous amount, since I have to make a liter scale culture.
Is this concentration required? What is the minimal efficient
concentration to fully induce beta-galactosidase synthesis?

--
Daniele EUSEBE(Ecole Normale Supérieure-INSERM U293-Paris-France)
e-mail:deu...@wotan.ens.fr

[Dr. Duncan Clark]

In article <deusebe-0910...@cherubin.ens.fr>, =?iso-8859-
1?q?Dani=E8le_EUSEBE?= <deu...@wotan.ens.fr> writes

> I have to induce beta-galactosidase in a liquid culture of E.coli Y1089
>lysogen for a lambda gt11 phage containing a beta-galactosidase-fusion
>protein gene.
> I have read in a protocole to use 10 mM IPTG, which seems to me an
>enormous amount, since I have to make a liter scale culture.
> Is this concentration required? What is the minimal efficient
>concentration to fully induce beta-galactosidase synthesis?
>

We routinely use 0.5mM final concn. for pUC derivative expression
vectors but we have gone down to 0.1mM on some.

10mM is way too much.

Duncan
-----------------------------------------------------------------------------
My mind's made up. Don't confuse me with the facts!
Duncan Clark
DNAmp Ltd.

[Richard P. Grant]

deu...@wotan.ens.fr (Daničle EUSEBE) writes:
> I have to induce beta-galactosidase in a liquid culture of E.coli Y1089
> lysogen for a lambda gt11 phage containing a beta-galactosidase-fusion
> protein gene.
> I have read in a protocole to use 10 mM IPTG, which seems to me an
> enormous amount, since I have to make a liter scale culture.

Routinely we use 0.1 mM for the modified promoter (Tac?) in pGEX,
but you should titrate for your application. This is a general rule - always
titrate on a small scale when you do something new.

> Is this concentration required? What is the minimal efficient
> concentration to fully induce beta-galactosidase synthesis?
>

Isn't b-gal promoter heat-shockable, anyway?

Richard
>
--
Richard P. Grant MA DPhil rpg...@molbiol.ox.ac.uk
Nuffield Department of Obstetrics and Gynaecology, University of Oxford.
http://users.ox.ac.uk/~lady0266
No wonder I can't go to parties anymore.

[Steven Goldberg]

In article <deusebe-0910...@cherubin.ens.fr>,
deu...@wotan.ens.fr (Daničle EUSEBE) wrote:

> I have to induce beta-galactosidase in a liquid culture of E.coli Y1089
> lysogen for a lambda gt11 phage containing a beta-galactosidase-fusion
> protein gene.
> I have read in a protocole to use 10 mM IPTG, which seems to me an
> enormous amount, since I have to make a liter scale culture.

> Is this concentration required? What is the minimal efficient
> concentration to fully induce beta-galactosidase synthesis?
>

> --
> Daniele EUSEBE(Ecole Normale Supérieure-INSERM U293-Paris-France)
> e-mail:deu...@wotan.ens.fr

For expression of recombinant proteins off a plasmid, I try a range from
35-1000 uM to try to optimize expression; usually I don't need more than
100 uM and get very good expression at 35 uM. 10 mM does seem excessively
high.

Steve

[LouPass]

I routinely use 1 mM (vast excess) but I have titrated the tac promoter
down to less than 10 micromolar on several of my plasmids..

[Nathan Siemers]

Using the (powerful) T7/lac promoter system (novagen vectors) for
driving expression of recombinant proteins in coli, I have found that
induction even at 100 micromolar IPTG is often toxic. As other people
have mentioned, titrate down in small scale trials. The clearest way
to determine toxicity due to induction that I have found is to induce
at innoculation of the culture, then follow the growth profile over
6-12 hours.

nathan

--
Nathan O. Siemers Bristol-Myers Squibb Pharmaceutical Research Institute
3005 First Avenue, Seattle, WA 98121 (206) 727-3741 sie...@bms.com