What about adding Glucose to the medium? It should lead to
suppression of the lactose operon. For induction, add IPTG.
> Date: Wed, 17 Mar 1999 10:31:12 +0100
> From: Cornelius Krasel <krasel at wpxx02.toxi.uni-wuerzburg.de>
> Reply-to: Cornelius Krasel <krasel at wpxx02.toxi.uni-wuerzburg.de>
> To: "bionet.molbio.methds-reagnts mail newsgroup" <bionet-news at dl.ac.uk>
> Subject: Re: protein expression in PLysS
> "kandasamy ravi" <ravi77 at MAILCITY.COM> wrote:
> > I have cloned my gene in pET15b transformed in to DH5 and isolated
> > DNA and retransformed in to pLysS BL21(DE3).checked the
> > sequences(it is in right frame) But i don't get overexpression
> > just little increase in expression.and the uninduced one is
> > expressing as good as induced.I tried different IPTG concentration
> > but no use and even the story is same in C43/C41 cell lines.
>> I remember that someone on this list reported previously that he ran
> into a similar problem. Finally he found out that his LB contained
> lactose. So, one possibility would be to obtain a different batch of
> peptone/yeast extract or alternatively use a defined medium for
> growing the bacteria.
>> You could also try to switch to pLysE cells instead of pLysS, but if
> you can already see expression of your construct without IPTG, I
> somewhat doubt that leakyness of the T7 promoter is the problem.
> /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher
> Str. 9 */ /* D-97078 Wuerzburg, Germany email:
>phak004 at rzbox.uni-wuerzburg.de SP4 */ /* "Science is the game we
> play with God to find out what His rules are." */
usual disclaimers apply * This message is RNAse free - please don't touch!
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de * wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
*unsolicited mail is *NOT* appreciated