How to get rid of Tn10 ?

Richard_Heath heath at mbcf.stjude.org
Thu Aug 10 19:15:56 EST 1995


In article <40ben1$ao4 at ccshst05.cs.uoguelph.ca>, speigham at uoguelph.ca (Seyed Mostafa Peighambari) writes:
> 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

The reference I have for this is Bochner et al, J. Bacteriol, 143, 926-930 
(1980), which uses fusaric acid and CTC (chlorotetracyline) (I think is the
correct ref, I can't find it right now to check).  We grow an overnight culture
of cells in rich medium, *dilute it 100-fold*, then plate onto the CTC plates
(0.1 ml per plate - do several plates for each strain!). Growth takes 36-48 
hours.  Individual colonies are then streaked for singles onto fresh CTC plates
(10, 20 or more colonies; growth should occur overnight now), and *then* scored 
for the loss of tetracycline resistance. In my hands I have got >80 % tet-s
each time.

Note:  apparently the density of the cells before the first CTC plating is
critical, so they should be diluted (I haven't tried it without!), and they
should also be re-streaked for singles before scoring.

If you need more details, please feel free to drop me an e-mail.

Good Luck!

Richard Heath,  richard.heath at stjude.org



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