?: avoid viscous bacterial culture

Ted M. tedm at darkwing.uoregon.edu
Thu Sep 5 22:25:40 EST 1996


In article <199609021035.TAA03946 at sutnfs.rs.noda.sut.ac.jp>,
ohba at rs.noda.sut.ac.jp (Hiroyoshi Ohba) wrote:

> Dear Netters;
> 
> XL1-Blue which was transformed with pBluescript derived vector got so
> viscous when cultured that cell was hardly separated from culture medium. 
> I guess that gummy material is mucous polysaccharide.  I used Super Broth
> (1% MOPS pH7.4, 3% bacto-tryptone, 2% yeast extract) supplimented with 1mM
> IPTG and 50 microgram/ml ampicilin and the culture was maintained at 30
> degree.  Possibly rich medium has induced production of nongrateful
> material.  This is not caused by the vector itself, but due to expression
> of inserted DNA because same vector with other insert did not make culture
> sticky.  How can I separate E. coli from viscous culture medium, or avoid
> viscous culture, or remove or digest viscous staff from the culture?  Thank
> you for your help.
> 
> regards,
> 
> ### ### ## ##  #  #   #    #     #    #    #   #  #  ## ## ### ###
> Hiroyoshi Ohba, Ph.D.


Hiroyoshi,
   I have a hard time understanding why your expressed protein would
induce polysaccharide formation, some lon(-) strains (BL21) form mucoid
colonies, but XL1Blue isn't one, is it? These cultures never go gooey
unless you have wholesale lysis and the goo is the cellular DNA filling
the culture. I have seen this when the Amp conc is off by 100 fold ( an
undergrad, really!), the pellet is sloppy, as well. Possibly the gene
product is a super lysozyme?
Alternatively there may be a contaminant in the transformed glycerol
stock. Try plating the cells on selective and unselective media before and
after induction, this will show if induction is killing everything. Also
look for foreign colonies. If your protein is a terrific antibiotic,
patent it and look into a yeast expression system!  Good Luck.

Ted Michelini
University of Oregon



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