E. coli restriction systems

"Henry Keith Reding ", MicroBio keithr at LENTI.MED.UMN.EDU
Tue Sep 27 16:35:04 EST 1994


Hopefully someone can help with this problem.  I believe we have a 
restriction problem with putting Clostridium acetobutylicum DNA into
any strain of E. coli.  We are trying to make a plasmid genomic DNA
library of C. acetobutylicum in E. coli using a shuttle vector consisting
of a pUC-pIM13 (from Staph.) vector.  This shuttle vector is necessary
so the library can be made in E. coli and then transformed into 
C. acetobutylicum mutants.  We are looking for regulatory genes in
C. acetobutylicum.  The pIM13 portion of the shuttle vector is required
for a Gram+ ori and the erythromycin resistance marker.

I have had no problems constructing a C. acetobutylicum library in E.
coli using Lambda ZAPII but this does not allow us to transform 
C. aceto. mutant with pools from the library in hope to select revertants.
When I try to make a plasmid library using this shuttle vector or just
a pUC vector, such as pUC19 or pBluescript, all of the inserts are chopped
up into pieces of 1.5 kb or less.  Often they appear the same size at 
about 1.5 kb.  Occasionally we can get 4-5 kb inserts cloned but this
rare and the number of transformants is low.  I have tried a variety
of hsd- and mcr- E. coli strains without success.  Futhermore, we transformed
C. aceto with one of the clones from E. coli, and then back transformed
from C. aceto into E. coli.  This resulted in the insert getting cut out
of the vector.

My questions are as follows:

1.  Why can the library be made easily in lambda but not in plasmids?  Is 
the DNA more accessible to restrictions systems when transformed instead
of transfected?

2.  Should I try building the library in Bacillus subtilis or Stapholcoccus
aureus instead of E. coli.

3.  Are other E. coli strains available that are further deleted in 
restrictions systems?

All thoughts are appreciated.

Keith Reding, PhD
USDA (formerly University of Minnesota, where is project is)



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