Are you adding vitamins to your M9 minimal? RosettaBlue is thiamin
requiring and can not grow in the absence of thiamin. The thiamin
requirement is so low that you can often get slow growth to a low OD
based on residual thiamin in the cells, but you will not get robust
growth.
Also, minimal recipes vary pretty drastically from one another, your
system may prefer one of the other recipes out there. Personally, I
have never really liked the standard M9 minimal recipe.
Contrary to another poster, I found that my cells tended to grow
better in minimal medium if I pre-adapted them to minimal by growing
my starters in the same minimal that I intended to use (with Met
instead of SeMet). However, I prefer to stick with high dilutions
(1:1000-1:100) so that may be the difference.
For years I used the MM/CA recipe from Pryor and Leiting, Protein
Expression and Purification, 1997 v10 pg 309. This recipe works well
and can be adapted for SeMet growth by subbing out the casamino acids
for a mixture of amino acids.
For the past few years I've been using the autoinduction recipes from
Studier, Protein Expression and Purification, v41 pg 207. I have
found that these work fantastically well (as long as you don't need
to express at really reduced temperature...I only use them down to 20°
C). There are recipes for SeMet incorporation in there as well.
Best of luck,
Cynthia
On Apr 18, 2007, at 10:34 PM, [EMAIL PROTECTED] wrote:
Hello everybody,
Sorry for an offtopic question. I am trying to express a protein
in M9
minimal media for Selenomet incorporation. When grown in LB this
protein
expressed very well and got good crystals. Diffraction was upto 2
A. I am
having a hard time expressing the same protein in Minimal media.
It took
nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with
IPTG in
minimal media and eventually got no protein expression. It looks
like the
cells are not growing or taking very long to grow. The cell line I am
using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen).
It expressed poorly in BL21 (DE3) when grown in LB and thus decided
to use
RosettaBlue (DE3). It worked very well in LB, but having a hard time
while expresing the same in minimal media using Rosetta Blue. Has
anybody
tried expression in minimal media using Rosetta Blue cell line? I am
planning to try overnight induction.
Any suggestions would be greatly appreciated.
Thanks,
Manish
*************************************************
Manish B. Shah, PhD.
Postdoctoral Fellow
Hauptman-Woodward Medical Research Institute
700 Ellicott Street
Buffalo, NY 14203.
*************************************************
____________________
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844
