In article <01HLR6J0LXKQ000CXC at hal.hahnemann.edu> MCGEED at HAL.HAHNEMANN.EDU
writes:
>Fellow micronetters, other than ethidium bromide, how does one cure a strain of
>its plasmids, when the plasmids lack antibiotic resistance markers to start
>with?
>Your help is very much appreciated.
>David J. McGee
>MCGEED at hal.hahnemann.edu
Several colleagues, including myself, have used Novobiocin successfully to
cure plasmids without any scorable marker. Novobiocin interferes with gyrase
(as does Nalidixic acid and several other agents - Oxolinic Acid/Coumermycin?)
which is needed for plasmid replication. In the 3 or 4 successful
applications I am most familiar with, the culture was transferred several
times near the MIC in broth for Novobiocin (determined from growth curves in
relation to control w/o Novob.). After several transfers in broth w/Novob.,
the cells were streaked/plated and individual colonies examined by miniprep
plasmid assay. When it worked, it apparently works well - nearly 50% or more
curing rate; but in other instances, it also didn't work for me. You may want
to consider using the plasmid as a probe against colony lifts **after** some
type of curing regimen (gyrase interfering agents, DNA intercalating agents,
growth at elevated temps., nutrient starvation/minimal medium, etc.) -
negative signal = cured cells.
Regards, Peter
p.s. - another approach I tried out of desperation was to clone an antibiotic
resistance marker into the plasmid and transform the wild-type strain - the
antibiotic selection caused loss of the wild-type plasmid for maintenance of
the abc/r-construct because of the same origin of replication(?) Anyway, I
then had a labelled wild-type plasmid with a scorable marker to follow during
the subsequent curing attempts. This worked very very well in booting out the
original plasmid (for the new construct).
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