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slimy cell paste of recombinant e.coli


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#1 biotef

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Posted 06 November 2017 - 10:23 PM

Slimy cell paste of E.coli?
We are doing fermentation for recombinant protein using E.coli culture. Earlier we used to obtain compact cell pellet on harvesting. But recently we observed that the cell pellet harvested are of slimy consistency and they do not homogenise in the washing buffer (tris-nacl-edta). We observe thread like consistency and no matter of stirring it do we get a homogeneous suspension.
 
Can someone guide me what is the reason for such phenomenon.
 
Thanks 
 
megha

 



#2 bob1

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Posted 07 November 2017 - 08:46 AM

It sounds like you have managed to either turned on biofilm formation (changes in medium??) or contaminated with a strain that produces a biofilm or capsule. I would streak some out on a plate and have a close look at the colonies formed.

 

It maybe that you need to go back and re-isolate the desired type (re-transform maybe), do some serious cleaning of all equipment involved in the culture, and check the materials used in the fermentation - perhaps you have a new supplier or degraded starting materials



#3 biotef

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Posted 07 November 2017 - 11:49 PM

we check the fermentation broth during the process, but there is no contamination observed.



#4 labtastic

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Posted 09 November 2017 - 09:21 AM

Your cells are lysing. The stringy-ness is the DNA released by lysed cells. 

 

This can be caused by a variety of things...toxic protein, detergent in the media, over growth, excessive flask shaking....or if you're using BL21 (DE3) derivatives there is a (very) remote possibility that prophage DNA is being triggered to form bacteriophage and lysing your cells, and so now you're contaminating everything with phage. You can avoid this by extensive cleaning and sterilization and subsequently using non-phage based BL21 T7 expression systems like BL21ai or NEB's T7 Express cells.


Edited by labtastic, 09 November 2017 - 10:40 AM.





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