Sorry, but I accidently deleted your message and lost your address.
It's been about 13-15 years since I did any plasmid work, and I wasn't
much good at it when I was doing it.
I did some work on Vibrio parahaemolyticus plasmids and found that none
were evident when it was haemolytic, but some plasmids did "pop out" when it
became non-haemolytic through sub-culturing
(The paper was submitted years ago and was promptly rejected. I guess
the reviewers were more familiar with the reverse systems in other bugs, even
though something similar to my work has been reported with Vibrio cholera, a
very close relative of V.p.)
Years ago I also read of similar problems to yours where a plasmid was
put in and 3 plasmids appeared. I believe it is a "packaging" problem. If you
look at a lot of publications, you'll see an aberrant band of chromosomal DNA
at 10 kD that everyone ignores. If it was random shearing, it should be a
smear, not a band (bell curve distribution?) of DNA. I noticed that a lot of
plasmids are either in this range or a multiple of this m.w. and so I believe
that plasmid replication, lysogeny, excision, etc. is based on "packets" of
DNA that associate with the "necklace" of chromosomal DNA. The replicative
machinery just merrily accepts it as part of the DNA packaging. Trouble arises
when the plasmid is too big and is either treated like more than 1 packet or
has the extra bits ignored.
We had a graduate student from U. of Guelph give a talk here at DRES
(Medicine Hat, Alberta), and he had similar problems as you.
With regards to stability, I've noticed at least for the haemolysin
of V.p. grown in synthetic media, there is transitional adaptation. The bug is
happy, unfavourable conditions cause it to become unhappy and express the
haemolysin, after a number of subcultures a population emerges that is happy
in this medium and so refuses to produce haemolysin. I saw some correlation
with plasmid expression, but didn't do enough work to have any confidence in
this.
Another observation I had was that V.p. was "schizophrenic". I forget
the details. I think it was that there was a balance sheet between pili and
flagella. On agar it produced more flagella to get around and hence less pili,
in broths it didn't need as many flagella and produced more pili. So you'll
get different colony morphologies depending on the number of subcultures in
each. (I've seen the same effect in Neisseria publications)
In summary, it's been several years and I'm not an expert. Still, I
suggest you try:
- making the media more complex (serum, yeast extract) to stablize the growth
requirements
- doing less isolation and subculturings for purity
- grow your bug requirements on agar (Roux flasks)
Have I reached the one asking the help, and is the above any use?....John
