How to get rid of Tn10 ?

[Seyed Mostafa Peighambari]

Hi everybody,
I have two E. coli strains (O2 and O78, originally Tet sensitive), with 
a Tn10 insertion in their cya genes. I am trying to select for the loss 
of the Tn10 using the Tet sensitive meduim (containing 12.5 mg/ml Tet and 
2 mg/ml Fusaric acid) as previously described by Maloy and Nunn (J. 
bacteriology, 145:1110-1112, 1981). Unfortunately my efforts have not been
successful sofar. I plated 0.1 ml of a rich broth culture of above 
strains on Tet sensitive plates and incubated for 48 hours. Then more than
one hundred of colonies grown on Tet sensitive plates were spot-inoculated
on L agar plates containing Tet (15 mg/ml). All grew well which means they
have not lost the Tn10. A Tet-sensitive strian used as control on L agar+Tet
plate did not grow. I appreciate if anybody share his experience with me 
in this regard. Thanks in advance. 

Regards,

Mostafa