bacillus popilae transposon-repost

Peter Kolchinsky pk20 at
Thu Dec 14 23:29:43 EST 1995

I have been working for a while with a strain of Bacillus popiliae, 
trying to isolate (an separate) its three naturally occurring plasmids.  
I would like to study its smallest, most elusive plasmid but am having a 
hard time even seeing it on a gel.  I have tried a number of techniques 
including restricting an entire plasmid prep and ligating the pieces into 
a vector.  I then transformed E.coli with the recombinant vector and 
screened for the pieces.  I have no way of telling which plasmid a piece 
came from except to do a hybridization, but then of course I have to get 
detectable amounts of the dna to run on the gel.  My latest idea was to 
mutagenize B. popiliae with a suitable selectable transposon, isolate the 
plasmid dna, and transform E.coli with it, selecting for the transposon 
marker.  I foresee two problems with this and would like advice.  I have 
not seen any commercially available transposons for B.popiliae, yet there 
are plenty for other Bacillus species, including B.subtilis.  Are these 
transposons species-specific or can I just use any Bacillus 
transposon-vector?  Also, once I transformed E.coli with the mutagenized 
Bacillus plasmids, would the E.coli be able to recognize the Bacillus 
origin-of-replication and thus replicate the plasmids, or are the ORI 
sites between the two bacteria lines drastically different?  If they are 
different, then could I find a transposon that carried an E.coli ORI 
sites so that the mutagenized Bacillus plasmids could replicate in 
E.coli?  I would appreciate any information whatsoever on this subject.  
My email address is pk20 at

thank you,

Peter Kolchinsky

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