BL21(DE3) problems

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Fri Nov 26 11:04:18 EST 1999


In article <Pine.SOL.4.05.9911261513260.26276-100000 at granby>, Andy
Scotter <pcxajs at unix.ccc.nottingham.ac.uk> writes
>       Hello there, hiopefully someone out there can help me out...
>
>       We have just cloned a small insert (145bp) into pET42 and
>transformed BL21(DE3) cells with it.  They grow fine on plates containing
>LB, 1% Glucose and 15micrograms/ml Kanamycin LBGK), with a reasonable
>transformation efficiency.  However, I can't get a liquid culture growing
>using LBGK.  I've tried 2.5ml, 5ml and 10ml cultures in Universals  (i.e.
>plenty of air) and I'm incubating the culture at 37 degrees centigrade.  
>       Anyone got any ideas?
>
>Thanks in advance....
>
>Andy
>

Sounds like your construct is lethal. Fairly standard behaviour for T7
expression systems. 

So apart from searching the archives for the umpteen previous threads on
this over the last few years, try:

Reducing growth temperature to 20-25C or lower.

Use BL21(DE3) pLysE or pLysS as host. pLys plasmids basically mop up any
stray T7 RNA pol produced through them producing T7 lysozyme.

Avoid yeast extract in your growth media.

Don't grow overnight then try and induce, as you will probably have
selected for a non-expressing variant. Stabilise the construct first.

Have fun

Duncan

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Duncan Clark
DNAmp Ltd.
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