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Why are my E. coli cells not pellet-able?


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#1 Lydiayi

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Posted 25 February 2009 - 08:11 AM

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

#2 perneseblue

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Posted 25 February 2009 - 08:38 AM

change in cell density. Change in the number of cells in solution, are also possible alternatives.
May your PCR products be long, your protocols short and your boss on holiday

#3 Lydiayi

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Posted 25 February 2009 - 08:46 AM

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.

change in cell density. Change in the number of cells in solution, are also possible alternatives.



#4 perneseblue

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Posted 25 February 2009 - 11:10 AM

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.
May your PCR products be long, your protocols short and your boss on holiday

#5 HomeBrew

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Posted 25 February 2009 - 02:01 PM

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.

#6 Lydiayi

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Posted 26 February 2009 - 12:25 PM

Hey, HomeBrew,

That's very interesting! Can you give me the citation of your paper published on that?
Thank you for your input!

Lydia

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.



#7 HomeBrew

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Posted 26 February 2009 - 08:01 PM

See here.




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