Dear Jennifer,
Though I was unaware of the permease involvement as Ronald as pointed
out but genereally we have observed quite uniform distribution of
proteins in a bacterial (in form of Inclusion bodies under
microscope). If some differences arise I am not sure how can one
distinguish them between those caused by cells being at different
stages of life cycle and those caused by the reasons you are looking
for. As you must be aware of the fact that once induced with IPTG
cells nearly stop growth and the amount of protein each bacterium will
produce depends on it's phase of growth. ( Induction in mid log phase
gives higher protein). So from any sample population I expect some
differences!!!
Best of Luck,
S.Ballal
Indus Biotherapeutics Ltd.
Ahmedabad India
http://www.indusbio.co.innone at mail.utexas.edu (Joe Doe) wrote in message news:<none-2103031702280001 at dhcp-133-242.icmb.utexas.edu>...
> 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
