Loosing expression efficency

[One Night]

Hello everyone;
I have a very great problem:
I built a new expression vector derived from pET9 eand pT7.7
and cloned my cDNA for expression. The recombinant plasmid
was then inserted in the BL21 DE3 pLysS strain.
The first exp. test was very good (we can consider it as the 100% of
the exp. level ). I had not success in reproducing this result because:
the exp. level is very variable (if I make a plate from my glycerol
stock
and pick 4 colony from it I obtain 4 different exp. levels (let's say
25, 31
55, 60% in respect of the original). The second thing is that the
overall
exp level seem to decrese from the beginig of the test (now I have only
about 15%).
Anyone has an idea to explain these facts?
Note:
I tried everything like inducing and collecting at different times,
making again all the stock solutions, and so on.
The resistance is Kanamicyn (and Chloramphenicol for pLys cells)
but I don't think that the losing of plasmid is the problem because I
start every time from a fresh plate from the glycerol stock and never
let the cell to grow for a too long time.
Anyway is there a test to know if the cells had got the Kanamicyn
resistance and had loose the plasmid?
Thank you
dr. Gianluca Molla

molla at imiucca.csi.unimi.it