Hey,
I'm expressing a lipopolysaccharide biosynthetic enzyme in E. coli. This is a enzyme from C. burnetii chimerized with a short C-terminal sequence from its E. coli homolog. The recombinant protein expressed as cytosolic oluble protein in E. coli BL21(DE3). But interestingly, comparing to the wild-type C. burbetii enzyme, this protein seems to make the E. coli cells unable to pellet well.
Wild-type C. burnetii enzyme/BL21(DE3):
-- Spin down at 6500 g for 10 min after fermentation overnight.
-- Formed nice pellet in the centrifuge bottle.
C. burnetii enzyme hybridized with a E. coli tail/BL21(DE3):
-- Spin down at 6500 g for 10 min after fermentation overnight.
-- Cell looks unsticky to each other and can't pellet down.
This happened several times. What would make the E. coli cells non-sticky? A change in morphology? Changes on the outer membrane? Anyone has any thought?
Thanks,
Lydia
-Lydiayi-
change in cell density. Change in the number of cells in solution, are also possible alternatives.
-perneseblue-
Thanks. Lower density or higher density would make it non-sticky?
I grew the cells at 37'C overnight and obtained a reasonably high amount of cells from 500 mL LB/amp, compared to normal fermentation of cells expressing the wild-type protein.
perneseblue on Feb 25 2009, 11:38 AM said:
change in cell density. Change in the number of cells in solution, are also possible alternatives.
-Lydiayi-
The cell are being pelleted by a centrifuge. Stickiness / clumping... while a factor isn't the only one. The density of the cells and the number (thus the pellet size, after x time of centrifuging) also play a role.
You could try increasing the centrifuge speed. Or spin for longer.
-perneseblue-
We had a mutant that over-produced an exopolysaccharide. We could not, for the life of us, get this strain to pellet; this phenotype was actually how we found what proved to be a very interesting EPS locus and regulatory mechanism in our bug.
-HomeBrew-
Hey, HomeBrew,
That's very interesting! Can you give me the citation of your paper published on that?
Thank you for your input!
Lydia
HomeBrew on Feb 25 2009, 05:01 PM said:
We had a mutant that over-produced an exopolysaccharide. We could not, for the life of us, get this strain to pellet; this phenotype was actually how we found what proved to be a very interesting EPS locus and regulatory mechanism in our bug.
-Lydiayi-
