P1 lysogeny: detection and curing
rfwhittier at hotmail.com
Wed Aug 8 05:16:31 EST 2001
Fellow methods mavens,
I've been having inordinate difficulties with what would seem to be
a routine plasmid construction. Well, maybe not completely routine.
It has inverted repeats, several loxP sites and variants thereof, and
the target plasmid is about 12 Kbp. However, an intermediate construct
with the inverted repeat and one loxP site is stable in this strain.
The fun and games start when I try to insert a rather large piece of
DNA that contains several more wt and mutant loxP sites. It's a
directional insertion (Bgl II with BamH I and Sal I with Xho I) so
it should be a piece of cake. Restriction digests of resultant
plasmids reveal a menagerie of constructs, many with bands that are
not stochiometric. I tried transforming the other plasmid with the
many loxP sites into this strain, and obtained well-defined colones.
However, when they are picked into liquid media under selection (amp)
their growth peters out while the cultures are only faintly cloudy.
Argh, methinks this be a P1 lysogen, Maties. However, I'd rather not
name the well-known strain in this forum without more definitive
evidence. I'm stuck with using this strain, because I have to keep
the inverted repeat stable. Ergo, the following two questions:
1) What's the easiest way to demonstrate P1 lysogeny? Inability to
plate phage lambda?
2) How does one go about curing a P1 lysogen? I know that P1 replicates
as a plasmid and has a maintenance system that kills cells that lose
it. But I've heard of P1 lysogens being cured, so the method must be
established somewhere back in the pre-PubMed literature.
Thanks for any suggestions.
Amersham Pharmacia Biotech K.K.
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