E.coli dies. HELP!!!!!

Kaj Stenberg kstenber at cc.Helsinki.FI
Thu Jun 6 07:57:54 EST 1996


This will be slightly lengthy, but I will try to explain what checks I'v
already done.

Basic:
I have a plasmid (12 of them) with mutants of a protein. The plasmids
have kanR in them. I grow them in BL21(DE3)plysS, induce with 0.4mM 
IPTG at OD 0.8, harvest after 18h. These conditions are tested and
have worked before. (I know that the time is extreme, but believe me).
Two months ago the cells grew nicely.

Problem:
The cells die just before induction, at OD roughly 0.7 (0.7 -> 0.1 in 10
minutes). The pre-ymp grows nicely, and the cells grow nicely in the big
flasks until they die.

Tested:
- all plasmids retransformed into fresh host
- all plasmids grown in small volume -> western. No growth problem,
western shows correct bands
- (after checking notes, death typically after OD-measuuraments). Two 
parallell flasks, one meassured with usual dispenser. No differences in 
growth. 
- All flasks (4) die simultaneously. Pre-ymp 150ml grown, 25ml into each
flask (OD<0.1). OD in production flasks rise to 0.6, then down to zero.
The rest of the pre-ymp continues growing, no problems. Between
cultivations the flasks are Iodine-treated, washed and autoclaved.

 Ideas:
Obviously medium, temperature, shaking, light, pH, and so on are always
identical.
 
-first idea was accident. I think not.
-second idea was phage. I do not think so
-third idea was accelerating amounts of lysozyme in medium. But why does
the small volume flasks grow? (there are 10 big flasks in the lab, I do
not always use the same ones).

My mind is blocked. If anyone has any explanations/ideas I'll be happy.
I do not care about the reason right now, but i want my protein!
 



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