In article
<Pine.A41.4.44.0303201519190.174684-100000 at homer11.u.washington.edu>,
James Bassuk <bassuk at u.washington.edu> wrote:
> On Wed, 19 Mar 2003, jennifer wrote:
>> > If i overexpress some protein, in the cell population, some cells may have a
> > lot of that protein, some may have less. Can I use western to distinguish
> > between them?
> > Or is there any method to measure a specific protein's concentration in a
> > single bacterial cell?
> >
> > BTW, does anbody know that whether using IPTG as an inducer will cause a
> > problem like that?
> >
>> Can you please help me understand why you want to differentiate between
> individual cells? Recombinant cultures that respond to IPTG usually
> involve a T7lac operon -- is this your system?
>> As long as you use antibiotics to guard against wild-type overgrowth, and
> pay attention to good methodology (i.e. like picking a fresh colony to
> grow up), then IPTG induction will result in all recombinant cells
> responding in sync.
>> Jim
Actually, this is not true at all concentrations of inducer and host
strains. At sub saturating levels of inducer an all or none phenomenon is
exhibited - some cells are fully induced, others are completely
uninduced. This was first shown in 1957 ( PNAS 43, 553-566). This is
because lac Y (permease) is induced by IPTG and at subsaturating levels of
IPTG the first cells to be induced, make more permease which soaks up all
the IPTG in the cells that were induced early.
For calibrated response to IPTG you need to use a host strain with a
permease mutation so that IPTG only gets in via diffusion (and hence
homogeneous) and not via the permease.
Novagen sells a strain called "tuner" for this purpose.
Some recent references on this sort of phenomenon for lac/tac and ara BAD
(similar problem) are:
Khlebnikov and Keasling (2002) Biotechnol Prog 2002, 672-674.
Morgan Kiss et al., (2002) PNAS 99 7373-7377.
Roland Saldanha
