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 cornell.edu
thank you,
Peter Kolchinsky
