pVE6060 tn delivery vector

DAB dboyd at ccu.umanitoba.ca
Wed Dec 14 13:13:47 EST 1994


I've been trying to use E. Maguin's  thermosensitive transposon deleivery
vector pVE6060 (J. Bact. 174:5633-5638, 1992) in Streptococcus mutans.
Essentially the vectors ori of replication is active at 28-30 ¡C but shuts
down over 35¡C, the transposase protein is provided by its gene (with a
Lactococcus lactis promoter) which is part of the vector, and the
transposon is simply a 1.3 kb erythromycin cassette between the outside
ends from Tn10. You establish the vector in your bacteria at 30¡C, shift
the culture to 37¡C and hope for transposition. They have shown it works in
B. subtilis. I've got the vector in my strep strain at 30¡C but I can't
seem to get the tn to do its thing at 37¡C.  I've been getting some
regular-sized colonies which Southern blotting seems to indicate either
have free plasmid or the whole plasmid integrated. Everytime except once
that I've plated out cells at 37¡C I've gotten some of these regular-sized
plasmids on what looks like a background of very small colonies. These very
small colonies shouldn't be true background however because S. mutans is
killed easily at even 0.5 µg/ml erm and I use 5-10 µg/ml on the plates.
What is happeneing? Should you plate out cells directly from a 30¡C
culture? Should you shift your liquid culture to 37¡C for a time before
plating out aliquots? If so, how much time at 37¡C? What are these very
small colonies in the 'background'? Might the transposition function just
not be active in streps such that any erm resistant colonies simply have
the whole vector integrated ? I know that there is more conditionally
lethal vectors for insertional inactivation and perhaps transposon delivery
being developed or already out there. Does anybody have any names or
references for ones (especially for tn delivery) that might work in
Gram-positives especially S. mutans?
Thanks for all help, Dave Boyd, dboyd at ccu.umanitoba.ca



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