E. coli W3110 not accepting two plasmids

Ralf Morgenstern Ralf.Morgenstern at imm.ki.se
Wed Mar 22 04:10:23 EST 1995


We have a problem when trying to put multiple plasmids into the E. coli
host W 3110.
1) Making the bacteria competent and transforming with a ampr plasmid: no
problem. Growing these bacteria in amp medium: no problem.
2) Making the W 3110 containing the ampr plasmid competent and transforming
in either a chloramphenicol(r) or kanamycin(r) plasmid: no problem
(selecting on both antibiotics). 
But!:
3) Growing bacteria from either "double plasmid containing" bacteria
(amp/chl or amp/kana) in liquid media containg both antibiotics: not
possible. Although the "double plasmid containing" bacteria (amp/kana) did
grow in kanamycin media.

The amp(r) plasmid is an expression construct similar to the pet series
vector with both a T7 and lac promoter based on pSPORT.
The kana(r) plasmid contains a temperature inducible T7 RNA polymerase and
is based on pBR 322.
The chl(r) plasmid expresses lysozyme (plys SL).

It is very puzzling that the double  plasmid containing E. coli can grow on
the double antibiotic plates but not in the same liquid media (it appears
that amp(r) is selectively lost).
The amp(r) and chl(r) resistant plasmids can coexist in other E. coli hosts
(BL-21 (DE-3)).
Question: Are different pairs of plasmids incompatible in certain strains
of bacteria? Has anyone else encountered similar problems and (hopefully)
solved them.
-- 
Ralf Morgenstern, IMM, Karolinska Institutet, Box 210, S-17177, SWEDEN
Tlf. +46-8-7287574, Fax, +46-8-334467, E-mail: ralf.morgenstern at imm.ki.se



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